Kinesin-Related Genes from Diplomonad, Sponge, Amphioxus, and Cyclostomes: Divergence Pattern of Kinesin Family and Evolution of Giardial Membrane–Bounded Organella

Naoyuki Iwabe and Takashi Miyata

Department of Biophysics, Graduate School of Science, Kyoto University


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
To understand the question of whether divergence of eukaryotic genes by gene duplications and domain shufflings proceeded gradually or intermittently during evolution, we have cloned and sequenced Giardia lamblia cDNAs encoding kinesins and kinesin-related proteins and have obtained 13 kinesin-related cDNAs, some of which are likely homologs of vertebrate kinesins involved in vesicle transfer to ER, Golgi, and plasma membrane. A phylogenetic tree of the kinesin family revealed that most gene duplications that gave rise to different kinesin subfamilies with distinct functions have been completed before the earliest divergence of extant eukaryotes. This suggests that the complex endomembrane system has arisen very early in eukaryotic evolution, and the diminutive ER and Golgi apparatus recognized in the giardial cells, together with the absence of mitochondria, might be characters acquired secondarily during the evolution of parasitism. To understand the divergence pattern of the kinesin family in the lineage leading to vertebrates, seven more Unc104-related cDNAs have been cloned from sponge, amphioxus, hagfish, and lamprey. The divergence pattern of the animal Unc104/KIF1 subfamily is characterized by two active periods in gene duplication interrupted by a considerably long period of silence, instead of proceeding gradually: animals underwent extensive gene duplications before the parazoan-eumetazoan split. In the early evolution of vertebrates around the cyclostome-gnathostome split, further gene duplications occurred, by which a variety of genes with similar structures over the entire regions were generated. This pattern of divergence is similar to those of animal genes involved in cell-cell communication and developmental control.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
From phylogenetic analyses of several gene families involved in signal transduction and developmental control, which diverged specifically in animal lineages by gene duplications and domain shufflings, we previously showed that the pattern of gene diversification is characterized by two active periods of gene duplication, which are interrupted by considerably long periods of silence, instead of proceeding gradually (Hoshiyama et al. 1998Citation ; Koyanagi et al. 1998a,Citation 1998b;Citation Suga, Ono, and Miyata 1999Citation ; Suga et al. 1999a,Citation 1999b;Citation Ono et al. 1999Citation , 2000; Miyata and Suga 2001Citation for review). The first extensive diversification of genes occurred at a very early stage of animal evolution before the divergence of parazoans and eumetazoans. In this period, animals underwent extensive gene duplications and domain shufflings that gave rise to different subfamilies with diverse functions, and almost complete sets of present-day subfamilies had been established. After the divergence of cephalochordates and vertebrates, the multiplicity of members in the same subfamily increased in the early evolution of vertebrates around or just before the cyclostome-gnathostome split by further gene duplications or genome wide duplications (or both) (Suga et al. 1999a;Citation Ono et al. 2000; Miyata and Suga 2001Citation for review). There is, however, no direct evidence to date for the question of whether gene diversification proceeded intermittently or gradually during the evolution of unicellular eukaryotes.

To clarify this problem, we have conducted cloning of cDNAs encoding the kinesins and kinesin-related proteins (KRPs) from Giardia lamblia, the most primitive protist that represents the earliest branching among extant eukaryotes in small subunit rRNA- and protein-based phylogenetic trees (Sogin et al. 1989Citation ; Hashimoto et al. 1994Citation , 1995Citation ). Including kinesin sequences from other eukaryotes reported to date, these sequences have been subjected to phylogenetic analyses of the kinesin family. The kinesins and KRPs are a large family of microtubule-motor proteins that share an ~340–450 amino acid motor domain (Moore and Endow 1996Citation ; Vale and Fletterick 1997Citation ; Block 1998Citation ). They serve as traffic regulators within cells for the transport of vesicles and organella (Moore and Endow 1996Citation ; Hirokawa 1998Citation ). They are also involved in cell division (Vale and Goldstein 1990Citation ; Walczak and Mitchison 1996Citation , Endow 1999Citation for reviews) and pattern formation in developing embryos (Moore and Endow 1996Citation ; Robb et al. 1996Citation ; Lane and Allan 1999Citation ). At least nine different subfamilies with distinct structures and functions belonging to the kinesin family have been identified to date (Moore and Endow, 1996Citation ; www.proweb.org/kinesin/index.html). Different subfamilies often differ in the organization of functional domains and thus differ in basic function, and members in the same subfamily are virtually identical in structure, motility properties, and cellular function (Moore and Endow 1996Citation ), but often differ in tissue distribution, and form a cluster in a family tree in most cases (e.g., Iwabe, Kuma, and Miyata 1996Citation ). The kinesin subfamilies might have arisen considerably early in eukaryotic evolution, at least before the divergence of animals, fungi, and plants (Moore and Endow 1996Citation ), as in other eukaryote-specific gene families (Iwabe, Kuma, and Miyata 1996Citation ).

To understand the pattern of gene diversification in early eukaryotes, we have conducted cloning of kinesin-related cDNAs from the diplomonad Giardia. We have obtained 13 giardial kinesin-related cDNAs, some of which are likely homologs of vertebrate kinesins involved in vesicle transfer to endoplasmic reticulum (ER), Golgi, and plasma membrane. From a phylogenetic analysis of the kinesin family, we report here that most, if not all, of the gene duplications that gave rise to different kinesin subfamilies have been completed before the earliest divergence of extant eukaryotes. Extensive subfamily-generating duplications in ancient times before the earliest branching among extant eukaryotes may have implications on the evolution of giardial membrane–bounded organella. A further phylogenetic analysis of the Unc104/KIF1 subfamily, including seven Unc104-related cDNA sequences from sponge, amphioxus, hagfish, and lamprey determined in this work, revealed two more clusters of gene duplications during animal evolution.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
Isolation and Sequencing of Giardial Kinesin–like Protein cDNAs
The extraction of total and poly(A)+ RNAs and genomic DNA of G. lamblia Polish strain was performed by standard techniques (Sambrook, Fritsch, and Maniatis 1989Citation , pp. 7.3–7.36, pp. 9.14–9.23). Giardia lamblia cDNAs were synthesized by reverse transcriptase (Super script II; GIBCO) using the oligo(dT) primer. These cDNAs or genomic DNAs were used as templates for PCR amplification with an Expand High Fidelity PCR System (Boehringer Mannheim). The sense and antisense degenerate primers were designed from conserved amino acid residues within the kinesin motor domain as follows: (1) 5'- TAGGTACCCARACNGGNACGGNAA-3' corresponding to the amino acid sequence QTGTGK, (2) 5'-TAGGATCCTTYGCNTAYGGNCARAC-3' corresponding to FAYGQT for sense primers, and (3) 5'-TAGAATTCTCISWICCIGCIARRTC-3' corresponding to DLAGSE for the antisense primer. Each primer contains BamHI or EcoRI restriction sites at the 5'-end (italicized). The first round of PCR amplification with primers 1 and 3 was conducted as follows: 2-min denaturation step at 94°C; then five cycles of 94°C (1 min), 46°C, 48°C or 52°C (2 min), and 72°C (5 min); followed by 30 cycles of 94°C (30 s), 60°C (1 min), and 72°C (2 min); and finally one cycle of 60°C (5 min) and 72°C (10 min). The second round of PCR with nested primers 2 and 3 was carried out with primary PCR products. The PCR products were separated in a 1.5% agarose gel containing ethidium bromide. Products of expected size were isolated as gel slices, purified using Gene Clean II (Bio101), and cloned into the pT7Blue vector (Novagen). Then, the Escherichia coli strain DH5{alpha} (TOYOBO) was transformed with a ligated vector. More than three independent clones were isolated and sequenced by the dideoxy chain termination method using synthetic oligonucleotide as primers. 3' regions of G. lamblia Klp cDNAs were obtained by 3' rapid amplification of cDNA ends (Gibco-BRL).

Isolation and Sequencing of Sponge, Lancelet, Hagfish, and Lamprey Unc104/KIF1-like cDNAs
Poly (A)+ RNA of Ephydatia fluviatilis (freshwater sponge) was extracted from the cells hatched from the gemmules (Seimiya et al. 1994) using the Quick Prep mRNA isolation kit (Pharmacia). Poly (A)+ RNA of Branchiostoma bercheri (lancelet) was extracted from the whole body. Poly (A)+ RNAs of Eptatretus burgeri (hagfish) and Lampetra reissneri (lamprey) were extracted from each brain. Cloning and sequencing strategies and primers used are the same as those in the case of G. lamblia. Unc104-specific antisense primers were also designed as follows: (4) 5'-TAGAATTCKYRTNGGRTCYTCRTT-3' corresponding to the amino acid sequence NEDPN(AN) and (5) 5'-TAGAATTCARYTTYTSYTCCCANGT-3' corresponding to the amino acid sequence TWE(EQ)KL.

Sequence Data
Members of the kinesin family were searched and identified in DDBJ release 46 by FASTA (Pearson and Lipman 1988Citation ) using the amino acid sequence of mouse MmKIF5a motor domain as a probe. Table 1 shows the sequence data used for inferring phylogenetic trees.


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Table 1 List of Sequence Data Used in the Present Analyses

 
Sequence Alignment
Multiple alignment of the amino acid sequences of kinesins and KRPs was carried out by a method developed recently by us (Katoh et al., 2002). This method is basically an extended version of the progressive approach of Feng and Doolittle (1987)Citation by improving the scoring system of amino acid substitutions and the calculation procedure of dynamic programming (Needleman and Wunsch 1970Citation ), by which the speed of computation has been greatly improved without sacrificing accuracy. The computation time required by the new method is only about one-tenth of that required by the standard method (for example, CLUSTAL W [Thompson, Higgins, and Gibson 1994Citation ], a widely distributed multiple alignment program).

Phylogenetic Tree Inference
The phylogenetic tree of the kinesin family was inferred by the weighted Neighbor-Joining (WNJ) method (Bruno, Socci, and Halpern 2000Citation ) and a recently developed heuristic approach, the genetic algorithm-based maximum likelihood method that outputs multiple trees, together with the best one (GAML-mt) (Katoh, Kuma, and Miyata 2001Citation ). One hundred initial trees were inferred by the WNJ method, on the basis of a set of alignments generated by bootstrap resamplings and distance matrices calculated by the maximum likelihood (ML) method under the Jones-Taylor-Thornton (JTT) model (Adachi and Hasegawa 1996Citation ). The evolutionary rate heterogeneity among sites was taken into account, assuming Yang's discrete gamma model (Yang 1994Citation ) with four rate categories and the optimized shape parameter, {alpha}. The actual computation was performed on a PC cluster composed of 32 Pentium III 500 MHz processors. In the GAML-mt method, the reliability index, and the bootstrap probability at each branching node of lineages were calculated. The reliability index at each branch node represents the difference {Delta} (= (Lmax - Lm)/{sigma}, where {sigma} represents the standard error) between the log-likelihood value (Lmax) of the ML tree and that (Lm) of tree m, giving the smallest {Delta} value among trees that do not support the clustering of the two lineages at the node. The bootstrap probability was calculated by the RELL method (Kishino, Miyata, and Hasegawa 1990Citation ), using tree topologies with the difference of log-likelihood values {Delta}Li (= Lmax - Li) less than {sigma}.


    Results and Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
Kinesin-Related cDNAs from G. lamblia
To understand the divergence pattern of genes that are recognized in all eukaryotes, we have conducted cloning and sequencing of kinesin-related cDNAs from the earliest diverging protist G. lamblia by the method described in the Materials and Methods. We have obtained 13 different giardial cDNAs encoding KRPs. Each of these cDNAs contains regions from the ATP-binding consensus sequence to poly(A) and encodes about 73% (~250 amino acids) of the motor domain. These sequences have been aligned with those of known members of the kinesin family for the motor domain. According to the alignment, the amino acid sequences of these cDNAs contain the kinesin signature motifs SSRSH and DLAGSE (Goldstein 1993Citation ; Bloom and Endow 1994Citation ) at the precise position. These cDNAs contain regions of 1321–3858 nucleotides which overlap with DNA fragments reported by the Giardia Genome Project (www.mbl.edu/baypaul/Giardia-HTML/index2.html), and their nucleotide sequences coincide in these fragments within minor sequence errors. In addition, as will be shown later, the amino acid sequences coded for by these cDNAs are phylogentically closely related to known kinesins and KRPs. It is therefore highly likely that the isolated cDNAs are the products of kinesin and kinesin-related genes.

Phylogenetic Tree of the Kinesin Family and Divergence of Subfamilies in the Early Evolution of Eukaryotes
On the basis of the alignment of kinesin motor domains, a phylogenetic tree of the kinesin family has been inferred by the WNJ method. For phylogenetic analysis, 142 kinesin sequences have been accumulated from the databases. Thirteen KRP cDNAs of G. lamblia and seven Unc104-related cDNAs of four metazoan species, E. fluviatilis (fresh water sponge), B. belcheri (lancelet), E. burgeri (hagfish), and L. reissneri (lamprey), determined in the present work (table 1 ) have also been included in the analysis. This data set covers most of the sequences used in recent tree inference by Endow's group (www.proweb.org/kinesin/index.html). To minimize the number of OTUs, 100 sequences were selected, excluding closely related sequences, genes diverged in vertebrate lineages, and rapidly evolving six OTUs (Giardia GlKlp12, yeast ScSmy1, Leishmania LmMCAK2, Leishmania L4768.04, yeast SpAC144.14, and yeast SpoBC2D10.21c) based on the preliminarily inferred trees. On the basis of the aforementioned set of sequence data selected, a phylogenetic tree was inferred by the WNJ method, using the distance matrix calculated by the "M method" under the JTT model (Adachi and Hasegawa 1996Citation ). The evolutionary rate variation among sites was included by applying the gamma distribution with {alpha} value 0.56 (fig. 1 ).



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Fig. 1.—Unrooted tree of the kinesin family. From a comparison of the kinesin motor domain sequences of fragment length (171 amino acids), the tree was inferred by the WNJ method. The evolutionary rate variation among sites was corrected by assuming the gamma distribution with {alpha} value 0.56. Gap sites were excluded from the analysis. The clusters corresponding to kinesin subfamilies are shaded. The classification of subfamilies and their names followed are as in The Kinesin Home Page (www.proweb.org/kinesin/index.html). Giardia lamblia kinesin-related cDNAs cloned in the present work are boxed. The number at each branch node represents the bootstrap probability obtained by 100 bootstrap resamplings (Felsenstein 1985Citation ) and the bootstrap probabilities higher than 50% are shown. Double circles, divergence of G. lamblia and other eukaryotes; rhombi, gene duplications; gene duplications that antedate the divergence of G. lamblia and other eukaryotes are shown by double rhombi or filled rhombi; stars, Saccharomyces kinesins or KRPs

 
As figure 1 shows, there is an excellent relationship between the clustering pattern of members in the family tree and structural and functional classification, as was already pointed out (Goodson, Kang, and Endow 1994Citation ; Moore and Endow 1996Citation ). Judging from the phylogenetic position, the cloned giardial GlKlp13, GlKlp11, GlKlp6, GlKlp2/GlKlp3, GlKlp8, and GlKlp7 are likely to belong to MCAK/KIF2, BimC, Unc104/KIF1, Krp85/95, C-terminal motor, and KHC subfamilies, respectively. Particularly, the giardial GlKlp8 and GlKlp13 show close resemblance in the structure of the C-terminal regions to the C-terminal motor, and MCAK/KIF2 subfamilies, respectively. In addition, the C-terminal regions of giardial GlKlp2/GlKlp3 are distantly related to the corresponding regions of the members of Krp85/95 subfamily, and the C-terminal region of GlKlp6 shows sequence similarity to Unc104/KIF1 in part. We failed to isolate giardial cDNAs homologous to three subfamilies (MKLP1, Kip3, and Chromokinesin/KIF4) comprising small numbers of animal or fungal members alone.

Judging from the phylogenetic positions of giardial kinesins and KRPs, the phylogentic tree of the kinesin family strongly suggests that most gene duplications that gave rise to different subfamilies antedate the divergence of G. lamblia and other eukaryotes, the earliest divergence among extant eukaryotes (marked by double circles in fig. 1 ). The nine subfamilies, MCAK/KIF2, Kip3, BimC, Chromokinesin/KIF4, Unc104/KIF1, KRP85/95, MKLP1, C-Terminal Motor, and KHC, have already been characterized fully by structural, functional, and phylogenetic analyses (Moore and Endow 1996Citation ; www.proweb.org/kinesin/index.html). Gene duplications that gave rise to these nine subfamilies are marked by double rhombi in figure 1 . According to the tree of figure 1 , the number Nb (Na) of these gene duplications that took place before (after) the divergence of Giardia and other eukaryotes is estimated to be 17 (0). To obtain a reliable estimate for Nb and Na based on a statistically solid background (Suga et al. 1997Citation ), 20 different phylogenetic trees of the kinesin family were inferred using 20 different sets of aligned sequences, each of which was generated by the standard bootstrap resampling procedure (Felsenstein 1985Citation ). The average values and the standard errors of Nb and Na were 15.6 ± 1.6, and 0.9 ± 0.7, respectively.

Using the alignment of selected OTUs, the phylogenetic tree of the 11 subfamilies has also been inferred by the GAML-mt method, a recently developed ML method as described in Materials and Methods. Evolutionary rate variations among sites were taken into account, assuming Yang's discrete gamma model (Yang 1994Citation ) with four rate categories and {alpha} value 0.65 (fig. 2 ). The ML tree supports the results obtained by WNJ analyses. All the subfamily-generating gene duplications antedate the divergence of Giardia and other eukaryotes. It is therefore highly likely that a basic set of kinesins and KRPs corresponding to the subfamilies have already been established in the very early stage of eukaryotic evolution before the earliest divergence of extant eukaryotes. Similar divergence patterns are also observed in rab, ADP-ribosylation factor, (ARF) and dynein families (Iwabe et al., unpublished data). Note that, as figure 1 shows, two such ancient gene duplications are identified within the C-terminal motor subfamily and two in the MCAK/KIF2 subfamily. It seems possible that each of the two subfamilies is classified into several independent subfamilies.



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Fig. 2.—Unrooted ML tree of the kinesin family. By sampling 32 OTUs from the alignment used in the WNJ tree, the tree was inferred by the ML method described in Materials and Methods; the evolutionary rate variation among sites was corrected by assuming the gamma distribution with {alpha} value 0.65; and gap sites were excluded from the comparison. The numbers at each branching node represent the reliability index estimated by the methods described in Materials and Methods. Double circles, divergence of G. lamblia and other eukaryotes; rhombi, gene duplications; gene duplications that antedate the divergence of G. lamblia and other eukaryotes are shown by double rhombi; stars, Saccharomyces kinesins or KRPs

 
Interestingly, S. serevisiae has only six kinesin and kinesin-related genes, as revealed by the complete genomic DNA sequence (Mewes et al. 1997Citation ). These S. serevisiae proteins (ScKar3, ScKip1, ScCin8, ScKip2, ScKip3, and ScSmy1, marked by "star" in fig. 1 ) are involved in spindle-chromosome motility and nuclear migration (Barton and Goldstein 1996Citation ; Lillie and Brown 1998Citation ; Miller et al. 1998Citation ; Endow 1999Citation ), but no organelle-vesicle transport kinesins exists on the genome. According to figure 1 , S. serevisiae ScKar3, ScKip1/ScCin8, and ScKip3 belong to C-terminal motor, BimC, and Kip3 subfamilies, respectively, and ScKip2 is probably a member of a group (human HsCENP-E, Ustilago UmKin1, and S. pombe SpoBC1604.20c). Because these subfamilies, together with other subfamilies in the kinesin family, are likely to have diverged in the very early evolution of eukaryotes before the separation of G. lamblia and other eukaryotes, as mentioned earlier, it is highly likely that selective deletions of kinesins involved in organelle-vesicle transport occurred on the lineage leading to extant S. cerevisiae during evolution. Why S. serevisiae lacks organelle-vesicle transport kinesins, most of which exist in primitive eukaryote G. lamblia with compact genome, remains to be clarified.

Parasitism as a Possible Mechanism for the Diminutiveness of the ER and Golgi Apparatus in the Giardial Cells
The parasitic protist G. lamblia is an evolutionarily problematic taxon. Giardia lamblia represents the earliest branching among extant eukaryotes in small subunit rRNA- and protein-based phylogenetic trees (Sogin et al. 1989Citation ; Hashimoto et al. 1994Citation , 1995Citation ). Remarkably, G. lamblia lacks many of membrane-bounded organella, including mitochondria, peroxisomes, typical ER and Golgi apparatus recognized in higher eukaryotic cells (Gillin, Reiner, and McCaffery 1996Citation ; Keeling 1998Citation ; Upcroft and Upcroft 1998Citation for reviews). These morphological characteristics of the giardial cell, together with the unique phylogenetic position, prompted one to consider that this organism may be a living fossil that remains in a primitive stage of evolution before acquisition of organella (Cavalier-Smith 1983Citation , 1987Citation ) and thus should provide deep insights into the early evolution of eukaryotes. Recent molecular phylogentic analyses, however, strongly suggest the secondary absence of mitochondria in G. lamblia (Hashimoto et al. 1998Citation ; Roger et al. 1998Citation ). Furthermore, evidence of the presence of diminutive Golgi apparatus (Lujan et al. 1995Citation ; Lanfredi-Rangel et al. 1999Citation ; Eichinger 2001Citation ) and ER (Soltys, Falah, and Gupta 1996Citation ) has recently been reported. Because no data was reported against the earliest divergence of Giardia lineage to date, these lines of evidence lead to a hypothesis that the lack of mitochondria, normal ER and Golgi apparatus in the giardial cell may be a result of evolution during parasitism, and the complex endomembrane system of sorting and transport, together with the nuclear envelope, have been established in the very early evolution of eukaryotes before the earliest divergence of extant eukaryotes (Alberts et al. 1994Citation , pp. 20; Lujan et al. 1995Citation ; Gillin, Reiner, and McCaffery 1996Citation ; Soltys, Falah, and Gupta 1996Citation ).

The present result that a basic set of kinesins and KRPs corresponding to the subfamilies have already been established in the very early stage of eukaryotic evolution before the separation of Giardia and other eukaryotes would provide an insight into this hypothesis on the diminutiveness of the ER and Golgi apparatus in the giardial cells. The kinesins and KRPs serve as traffic regulators within cells for the transport of vesicles and organella (Moore and Endow 1996Citation ; Hirokawa 1998Citation ). They are also involved in cell division (Vale and Goldstein 1990Citation ; Walczak and Mitchison 1996Citation ; Endow 1999Citation for reviews) and pattern formation in developing embryos (Moore and Endow 1996Citation ; Robb et al. 1996Citation ; Lane and Allan 1999Citation ). The early divergence of the basic set of these kinesins and KRPs suggests that the complex endomembrane system might have arisen in the very early evolution of eukaryotes before the earliest divergence of extant eukaryotes.

We recently found the presence of many overlapping genes in G. lamblia (Iwabe and Miyata 2001Citation ). In addition, no or few introns have been identified in the giardial genome (Smith et al. 1998Citation ; Upcroft and Upcroft 1998Citation ; Nixon et al. 2002Citation ). From these lines of evidence, we suggest that the diminutive ER and Golgi apparatus recognized in the giardial cells, together with the absence of mitochondria and the extremely economized usage of DNA sequence, might be characters acquired secondarily during the evolution of parasitism.

Divergence Pattern of Unc104/KIF1 Subfamily Members and Related Kinesins
As mentioned earlier, divergence of different subfamilies is very old, going back to dates before the earliest divergence among extant eukaryotes. According to the phylogenetic trees of the kinesin family reported to date, the multiplicity of members in the same subfamily increases in the later stage of evolution by further gene duplications (e.g., Moore and Endow 1996Citation ). From phylogenetic analyses of gene families involved in the signal transduction and developmental control that have diverged specifically in animal lineages, we recently showed that the pattern of gene diversification is characterized by two active periods in gene duplication interrupted by considerably long periods of silence, instead of proceeding gradually (Hoshiyama et al. 1998Citation ; Koyanagi et al. 1998a,Citation 1998b;Citation Suga, Ono, and Miyata 1999Citation ; Suga et al. 1999a,Citation 1999b;Citation Ono et al. 1999Citation , 2000; Miyata and Suga 2001Citation for review): In the very early stage of animal evolution before the parazoan-eumetazoan split, animals underwent extensive gene duplications that gave rise to different subfamilies with diverse functions, and almost complete sets of present-day subfamilies had been established within the period. After the divergence of cephalochordates and vertebrates, the multiplicity of members in the same subfamily rapidly increased in the early evolution of vertebrates around or just before the cyclostome-gnathostome split by further gene duplications (Suga et al. 1999a;Citation Ono et al. 2000; Miyata and Suga 2001Citation for review). It may be interesting to know whether the divergence pattern in each kinesin subfamily is similar to that found in animal-specific gene families.

To clarify this question, we have conducted cloning and sequencing of Unc104-related cDNAs from E. fluviatilis, a sponge, B. belcheri, an amphioxus, E. burgeri, a hagfish, and L. reissneri, a lamprey by the method described in Materials and Methods. We obtained two cDNAs (EfKlF1 and EfKlp2) from sponge, two cDNAs (EbKIF1A and EbKIF1B) from hagfish, two cDNAs (LrKIF1A and LrKIF1B) from lamprey, and one cDNA BbKIF1 from lancelet. From a comparison of these sequences with closely related sequences reported to date, phylogenetic trees have been inferred by a heuristic ML method described in Materials and Methods (fig. 3 ).



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Fig. 3.—ML tree of the Unc104/KIF1 subfamily. From a comparison of sequences of the kinesin motor domain and C-terminal domain of fragment lengths, the phylogenetic trees were inferred by the ML method described in Materials and Methods; the evolutionary rate variation among sites was corrected by assuming the gamma distribution with optimized shape parameter {alpha}; gap sites were excluded from the comparison. The numbers at each branching node represent the bootstrap probability-reliability index estimated by the methods described in Materials and Methods. Open circle, cyclostome-gnathostome split; filled circle, parazoan-eumetazoan split; gray circle, fungi (slime mold)-animal split, open rhombus, gene duplication that postdates the cephalochordate-vertebrate split; filled rhombus, gene duplication that antedates the parazoan-eumetazoan split; gray rhombus, gene duplication that antedates the deuterostomes-protostomes split. (a) Phylogenetic tree of animal KIF1 homologs, Dictyostelium, DdUnc104, and giardial Glklp6. The number N of residues compared is 215; {alpha} = 0.53. (b) Phylogenetic tree of the MmKIF1A/DmUnc104 subgroup. Five sequences HsRBKIN1, HsGAKIN, DmKlp73, CeF56E3.3, and EfKlp2 were used as an outgroup. N = 241; {alpha} = 0.32

 
According to figure 3a, Unc104 and the related kinesin (Unc104-RK) group are classified into five subgroups. These are MmKIF1A/DmUnc104, HsGAKIN/ DmKlp73, HsCMKrp/DmKlp38B, HsJ777L9/DmKlp98A, and CeLR144.1. The cloned sponge EfKlp2 belongs to HsGAKIN/DmKlp73. The phylogenetic tree (fig. 3a ) suggests that at least three groups out of five are likely to have diverged before the separation of sponge and other animals by gene duplication (the divergence times of the remaining two are unknown). In addition, these gene duplications postdate the divergence of animals and slime mold (fig. 3a ), although a part of the gene duplication antedates the animals-fungi split. It is therefore highly likely that the gene duplication that gave rise to the five subgroups occurred in the very early evolution of multicellular animals before the parazoans-eumetazoans split.

In the subgroup containing MmKIF1A/DmUnc104, two more gene duplications that gave rise to three different kinesins are observed (fig. 3b ). These gene duplications evidently postdate the divergence of cephalochordates and vertebrates, but the precise dates are uncertain because the branching orders of the seven vertebrate OTUs are obscure. This suggests that these gene duplications took place in the early evolution of vertebrates around or just before the divergence of cyclostomes and gnathostomes. This is consistent with the divergence patterns observed in the subfamilies of the protein tyrosine kinase (PTK) family (Suga et al. 1999bCitation ) and the protein tyrosine phosphatase (PTP) family (Ono-Koyanagi et al. 2000Citation ). It is therefore highly likely that the Unc104-RK group diverged specifically in animal lineages, and the divergence pattern is quite similar to those observed in animal gene families involved in the signal transduction. Similar divergence patterns would be observed in other kinesin subfamilies.

Thus, in the lineage leading to vertebrates, the divergence of the members of the kinesin family by gene duplications seems to have occurred intermittently, instead of proceeding gradually. The divergence pattern might be characterized by at least three active periods in gene duplication interrupted by considerably long periods of silence (fig. 4 ). The first active period corresponds to dates before the separation of G. lamblia and other eukaryotes, in which a variety of kinesin subfamilies which differ in basic function and domain organization has been generated by extensive gene duplications and domain shufflings. It seems likely that, since the separation from fungi and plants, early animals lived in a period before the parazoan-eumetazoan split might have undergone further gene duplications, by which the multiplicity of members in the same subfamily increased, as demonstrated by Unc104 and the related kinesin group. In the early evolution of vertebrates before the cyclostome-gnathostome split, further gene duplications have occurred, by which a variety of genes with similar structures over their entire regions had been established in each subfamily. Although it is not clear at present whether the divergence pattern observed in the Unc104-RK group is a general one commonly found in other kinesin subfamilies, the pattern in Unc104-RK is quite similar to those in animal-specific gene families. This scenario of stepwise gene diversification would be useful for tree-based classification of family members that have diverged during eukaryotic evolution.



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Fig. 4.—Intermittent divergence of the kinesin family in eukaryotic lineage leading to vertebrates. Circles I, II, and III are the periods in which extensive gene duplications occurred. In period I, basic kinesin genes corresponding to subfamilies with different structures and functions were created by gene duplications and domain shufflings. In the later periods II and III, the multiplicity of members in the same subfamily increased by further gene duplications

 

    Data Deposition
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
The nucleotide sequence data reported in this article have been deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases (accession numbers: AB028051AB028063 and AB070245AB070251).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 
We thank Prof. M. Hasegawa of the Institute of Mathematical Statistics, Drs. K. Kuma, H. Suga, D. Hoshiyama, and K. Katoh of Kyoto University for discussion and comments. We also thank Dr. Hashimoto of the Institute of Mathematical Statistics for kindly providing genomic DNA and total RNA of G. lamblia. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan.


    Footnotes
 
Naruya Saitou, Reviewing Editor

Keywords: gene duplication kinesin family phylogenetic tree endoplasmic reticulum Golgi apparatus parasitism Back

Address for correspondence and reprints: Takashi Miyata, Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502 Japan. E-mail: miyata{at}biophys.kyoto-u.ac.jp . Back


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Data Deposition
 Acknowledgements
 References
 

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Accepted for publication May 9, 2002.