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The tradition of cockfighting is widespread throughout the world. There is no doubt that the gamecock has evolved together with the human culture of cockfighting for a long time. In Japan, there is a group of gamecocks called "Shamo" that are used specifically for cockfighting. However, the process of the geographic distribution of cockfighting and the influx route of gamecocks into Japan are totally unclear. The molecular evolutionary study of gamecocks is obviously useful to gain profound insight into the understanding of not only the evolutionary origin of "Shamo" but also the distribution process of cockfighting as a culture. In this study, we collected blood samples of gamecocks from 11 different prefectures in Japan. Then, a phylogenetic tree was constructed using a total of 42 mtDNAs (D-loop, 1100 bp) sequenced. It showed that Japanese Shamo was clearly separated into two different groups: One group contains the samples from the island of Okinawa and the other group is composed of the samples mainly from Kyushu and Honshu of Japan. It suggests that Japanese Shamo must have been brought to Japan from two different origins. Our examination of historical records showed that the results of the phylogenetic analysis is consistent with the view that Japanese Shamo was originated from Southeast Asia and the mainland China independently, but was geographically a bit mixed afterwards.

Corynebacterium species are members of gram-positive bacteria closely related to Mycobacterium species, both of which are classified into the same taxonomic order Actinomycetales. Recently, three corynebacteria, Corynebacterium efficiens, Corynebacterium glutamicum, and Corynebacterium diphtheriae have been sequenced independently. We found that the order of orthologous genes in these species has been highly conserved though it has been disrupted in Mycobacterium species. This synteny suggests that corynebacteria have rarely undergone extensive genome rearrangements and have maintained ancestral genome structures even after the divergence of corynebacteria and mycobacteria. This is the first report that the genome structures have been conserved in free-living bacteria such as C. efficiens and C. glutamicum, although it has been reported that obligate parasites such as Mycoplasma and Chlamydia have the stable genomes. The comparison of recombinational repair systems among the three corynebacteria and Mycobacterium tuberculosis suggested that the absence of recBCD genes in corynebacteria be responsible for the suppression of genome shuffling in the species. The genome stability in Corynebacterium species will give us hints of the speciation mechanism with the non-shuffled genome, particularly the importance of horizontal gene transfer and nucleotide substitution in the genome.

We describe the current status of the gene expression database CIBEX (Center for Information Biology gene EXpression database, http://cibex.nig.ac.jp), with a data retrieval system in compliance with MIAME, a standard that the MGED Society has developed for comparing and data produced in microarray experiments at different laboratories worldwide. CIBEX serves as a public repository for a wide range of high-throughput experimental data in gene expression research, including microarray-based experiments measuring mRNA, serial analysis of gene expression (SAGE tags), and mass spectrometry proteomic data.

Corynebacterium efficiens is the closest relative of Corynebacterium glutamicum, a species widely used for the industrial production of amino acids. C. efficiens but not C. glutamicum can grow above 40 degrees C. We sequenced the complete C. efficiens genome to investigate the basis of its thermostability by comparing its genome with that of C. glutamicum. The difference in GC content between the species was reflected in codon usage and nucleotide substitutions. Our comparative genomic study clearly showed that there was tremendous bias in amino acid substitutions in all orthologous ORFs. Analysis of the direction of the amino acid substitutions suggested that three substitutions are important for the stability of the C. efficiens proteins: from lysine to arginine, serine to alanine, and serine to threonine. Our results strongly suggest that the accumulation of these three types of amino acid substitutions correlates with the acquisition of thermostability and is responsible for the greater GC content of C. efficiens.

Despite their high degree of genomic similarity, reminiscent of their relatively recent separation from each other ( approximately 6 million years ago), the molecular basis of traits unique to humans vs. their closest relative, the chimpanzee, is largely unknown. This report describes a large-scale single-contig comparison between human and chimpanzee genomes via the sequence analysis of almost one-half of the immunologically critical MHC. This 1,750,601-bp stretch of DNA, which encompasses the entire class I along with the telomeric part of the MHC class III regions, corresponds to an orthologous 1,870,955 bp of the human HLA region. Sequence analysis confirms the existence of a high degree of sequence similarity between the two species. However, and importantly, this 98.6% sequence identity drops to only 86.7% taking into account the multiple insertions/deletions (indels) dispersed throughout the region. This is functionally exemplified by a large deletion of 95 kb between the virtual locations of human MICA and MICB genes, which results in a single hybrid chimpanzee MIC gene, in a segment of the MHC genetically linked to species-specific handling of several viral infections (HIV/SIV, hepatitis B and C) as well as susceptibility to various autoimmune diseases. Finally, if generalized, these data suggest that evolution may have used the mechanistically more drastic indels instead of the more subtle single-nucleotide substitutions for shaping the recently emerged primate species.

Among the bilateral animals, a centralized nervous system is found in both the deuterostome and protostome. To address the question of whether the CNS was derived from a common ancestor of deuterostomes and protostomes, it is essential to know kinds of genes existed in the CNS of the putative common ancestor and to trace the evolutionary divergence of genes expressed in the CNS. To answer these questions, we took a comparative approach using different species, particularly focusing on one of the lower bilateral animals, the planarian (Platyhelminthes, Tricladida), which is known to possess a CNS. We determined the nucleotide sequence of ESTs from the head portion of planarians, obtaining 3,101 nonredundant EST clones. As a result of homology searches, we found that 116 clones had significant similarity to known genes related to the nervous system. Here, we compared these 116 planarian EST clones with all ORFs of the complete genome sequences of the human, fruit fly, and nematode, and showed that >95% of these 116 nervous system-related genes, including genes involved in brain or neural morphogenesis, were commonly shared among these organisms, thus providing evidence at the molecular level for the existence of a common ancestral CNS. Interestingly, we found that approximately 30% of planarian nervous system-related genes had homologous sequences in Arabidopsis and yeast, which do not possess a nervous system. This implies that the origin of nervous system-related genes greatly predated the emergence of the nervous system, and that these genes might have been recruited toward the nervous system.

The origin of the brain remains a challenging problem in evolutionary studies. To understand when and how the structural brain emerged, we analyzed the central nervous system (CNS) of a lower invertebrate, planarian. We conducted a large-scale screening of the head part-specific genes in the planarian by constructing a cDNA microarray. Competitive hybridization of cDNAs between a head portion and the other body portion of planarians revealed 205 genes with head part-specific spikes, including essential genes in the vertebrate nervous system. The expression patterns of the top 30 genes showing the strongest spikes implied that the planarian brain has undergone functional regionalization. We demonstrate the complex cytoarchitecture of the planarian brain, despite its simple superficiality of the morphology.

The insect Toll family of proteins and their mammalian counterparts seemingly shared one common ancestor and evolved independently. Here we demonstrated that the prototype of the mammalian-type (M-type) Toll family is shared by the fish and humans. According to the draft of the pufferfish Fugu genome project, the signature Toll-IL-1 receptor homology domain (TIR domain) has been conserved during evolution. FuguTLR2, 3, 5, 7, 8 and 9 members correspond structurally to respective mammalian TLRs. One Fugu TLR showed equally high amino acid identity to human TLR1, 6 and 10, and we named it FuguTLR1. Fugu rubripes has genes for TLR21 and 22, which are unique to fish. One possible interpretation of these findings is that TLR1, 2, 3, 4, 5, 7, 8, 9, 21 and 22 existed in the ancestral genome common to fish and mammals, and that TLR4 was lost in the fish lineage, while TLR21 and 22 were lost in the mammalian lineage. Strikingly, a solitary ascidian, Halocynthia roretzi, has only a few Toll-like proteins, which, like Caenorhabditis elegans Toll, represent primitive ones before the expansion of the Toll family. Therefore, the expansion of TLR genes should have occurred earlier than fish, but not C. intestinalis, separated evolutionarily from mammals. These results infer that the appearance of the M-type innate system was completed before or concomitant with the appearance of acquired immunity. We interpret the present data to mean that the differences of TLRs identified in this study between fishes and humans may be rather peripheral, partially due to selection pressure exerted by pathogens in distinct environments.

The study of planarian regeneration may help us to understand how we can rebuild organs and tissues after injury, disease or ageing. The robust regenerative abilities of planarians are based upon a population of totipotent stem cells (neoblasts), and among the organs regenerated by these animals is a well-organized central nervous system. In recent years, methodologies such as whole-mount in situ hybridizations and double-stranded RNA have been extended to planarians with the aim of unravelling the molecular basis of their regenerative capacities. Here we report the identification and characterization of nou-darake (ndk), a gene encoding a fibroblast growth factor receptor (FGFR)-like molecule specifically expressed in the head region of the planarian Dugesia japonica. Loss of function of ndk by RNA interference results in the induction of ectopic brain tissues throughout the body. This ectopic brain formation was suppressed by inhibition of two planarian FGFR homologues (FGFR1 and FGFR2). Additionally, ndk inhibits FGF signalling in Xenopus embryos. The data suggest that ndk may modulate FGF signalling in stem cells to restrict brain tissues to the head region of planarians.

In previous studies, we have shown that dorsoventral (DV) interaction evokes not only blastema formation, but also morphogenetic events similar to those that occur in regeneration. However, it is still unclear what kinds of signal molecules are involved in the DV interaction. To investigate the signal systems involved in the DV interaction, we focused on a noggin-like gene (Djnlg) identified by the planarian EST project. Djnlg is the first noggin homologue isolated from an invertebrate. In DjNLG, the positions of nine cysteine residues which may be essential for dimer formation were well conserved, but overall, the amino acid sequence of DjNLG did not show high similarity to the sequences of vertebrate Noggins. Expression of Djnlg was observed only in the proximal region of the branch structures in the brain of intact planarians, suggesting that Djnlg may have a role in pattern formation in the brain. Interestingly, transient strong expression of Djnlg was observed in the amputated region of regenerating planarians. Djnlg-expressing cells were detected beneath the muscle 9 h after amputation and were then detected in the ventral subepidermal region of the blastema. The induction of Djnlg expression by amputation was not affected by X-ray irradiation, even though the stem cells were completely eliminated, implying the existence of signal-producing cells which may provide a positional cue to the stem cells. In DV reversed grafting, expression of Djnlg was strongly induced in the DV boundary between the host and donor. These results suggest that ectopic DV interaction may induce expression of Djnlg in the positional cue-producing cells, and that it might be involved in stimulation of blastema formation as well as DV patterning of the body.

Planarians are attractive animals in which various questions related to the central nervous system (CNS) can be addressed, such as its origin and evolution, its degree of functional conservation among different organisms, and the plasticity and regenerative capabilities of neural cells and networks. However, it is first necessary to characterize at the gene expression level how this CNS is organized in intact animals. Previous studies have shown that the planarian brain can be divided into at least three distinct domains based on the expression of otd/Otx-related genes. In order to further characterize the planarian brain, we have recently isolated a large number of planarian neural-specific genes through DNA microarrays and ESTs projects. Here, we describe new molecular domains within the brain of intact planarians by the expression of 16 planarian neural-specific genes, including the putative homologues of protein tyrosine phosphatase receptor, synaptotagmin VII, slit, G protein and glutamate and acetylcholine receptors, by in situ hybridization in both whole-mount and transverse sections. Our results indicate that planarian otd/Otx-positive domains can be further subdivided into distinct molecular regions according to the expression of different neural genes. We found differences at the gene expression level between the dorsal and ventral sides of the brain, along its antero-posterior axis and also between the proximal and distal parts of the brain lateral branches. This high level of regionalization in the planarian brain contrasts with its apparent simplicity at the morphological level.

Background: The complete genomes of three animals have been sequenced by global research efforts: a nematode worm (Caenorhabditis elegans), an insect (Drosophila melanogaster), and a vertebrate (Homo sapiens). Remarkably, their relationships have yet to be clarified. The confusion concerns the enigmatic position of nematodes. Traditionally, nematodes have occupied a basal position, in part because they lack a true body cavity. However, the leading hypothesis now joins nematodes with arthropods in a molting clade, Ecdysozoa, based on data from several genes., Results: We tested the Ecdysozoa hypothesis with analyses of more than 100 nuclear protein alignments, under conditions that would expose biases, and found that it was not supported. Instead, we found significant support for the traditional hypothesis, Coelomata. Our result is robust to different rates of sequence change among genes and lineages, different numbers of taxa, and different species of nematodes., Conclusion: We conclude that insects (arthropods) are genetically and evolutionarily closer to humans than to nematode worms.

The planarian central nervous system (CNS) can be used as a model for studying neural regeneration in higher organisms. Despite its simple structure, recent studies have shown that the planarian CNS can be divided into several molecular and functional domains defined by the expression of different neural genes. Remarkably, a whole animal, including the molecularly complex CNS, can regenerate from a small piece of the planarian body. In this study, a collection of neural markers has been used to characterize at the molecular level how the planarian CNS is rebuilt. Planarian CNS is composed of an anterior brain and a pair of ventral nerve cords that are distinct and overlapping structures in the head region. During regeneration, 12 neural markers have been classified as early, mid-regeneration and late expression genes depending on when they are upregulated in the regenerative blastema. Interestingly, the results from this study show that the comparison of the expression patterns of different neural genes supports the view that at day one of regeneration, the new brain appears within the blastema, whereas the pre-existing ventral nerve cords remain in the old tissues. Three stages in planarian CNS regeneration are suggested.

Molluscs display a rich diversity of body plans ranging from the wormlike appearance of aplacophorans to the complex body plan of the cephalopods with highly developed sensory organs, a complex central nervous system, and cognitive abilities unrivaled among the invertebrates. The aim of the current study is to define molecular parameters relevant to the developmental evolution of cephalopods by using the sepiolid squid Euprymna scolopes as a model system. Using PCR-based approaches, we identified one anterior, one paralog group 3, five central, and two posterior group Hox genes. The deduced homeodomain sequences of the E. scolopes Hox cluster genes are most similar to known annelid, brachiopod, and nemertean Hox gene homeodomain sequences. Our results are consistent with the presence of a single Hox gene cluster in cephalopods. Our data also corroborate the proposed existence of a differentiated Hox gene cluster in the last common ancestor of Bilaterians. Furthermore, our phylogenetic analysis and in particular the identification of Post-1 and Post-2 homologs support the Lophotrochozoan clade.

Tyrosinase is the key enzyme for synthesizing melanin pigments, which primarily determine mammalian skin coloration. Considering the important roles of pigments in the evolution and the adaptation of vertebrates, phylogenetic changes in the coding and flanking regulatory sequences of the tyrosinase gene are particularly intriguing. We have now cloned cDNA encoding tyrosinase from Japanese quail and snapping turtle. These nonmammalian cDNA are highly homologous to those of the mouse and human tyrosinases, whereas the 5' flanking sequences are far less conserved except for a few short sequence motifs. Nevertheless, we demonstrate that the 5' flanking sequences from the quail or turtle tyrosinase genes are capable of directing the expression of a fused mouse tyrosinase cDNA when introduced into cultured mouse albino melanocytes. This experimental method, which reveals the functional conservation of regulatory sequences in one cell type (the melanocyte), may be utilized to evaluate phylogenetic differences in mechanisms controlling specific gene expression in many other types of cells. We also provide evidence that the 5' flanking sequences from these nonmammalian genes are functional in vivo by producing transgenic mice. Phylogenetic changes of vertebrate tyrosinase promoters and the possible involvement of conserved sequence motifs in melanocyte-specific expression of tyrosinase are discussed.

The Gobioidei is a large suborder in the order Perciformes and consists of more than 2000 species belonging to about 270 genera. The vast number of species and their morphological specialization adapted to diverse habits and habitats makes the classification of the gobioid fishes very difficult.A comprehensive estimation of the evolutionary scenario of all gobioid fishes using only morphological information is difficult for two major reasons: first, in addition to wide ecological diversification, there is a trend towards specialization and degeneration of morphological characters among these species; second, an appropriate outgroup of gobioid fishes has not been recognized. Based upon nucleotide sequence comparisons of gobioid mitochondrial cytochrome b genes, we established the phylogenetic relationships of their differentiation into many groups of morphological and ecological diversity. The phylogenetic trees obtained show that most species examined have diverged from each other almost simultaneously or during an extremely short period of time.

Tyrosinase is the key enzyme required for the synthesis of melanin pigments. Sequence comparison and functional analysis of the 5' upstream regions of vertebrate tyrosinase genes have revealed the importance of conserved E-box motifs in regulating their specific expression in pigment cells, optic cup-derived retinal pigment epithelium (RPE) and neural crest-derived melanocytes. In ascidians (more basal protochordates), two pigment cells that resemble vertebrate RPE cells are formed and specifically express the orthologous tyrosinase gene (HrTyr) in the cerebral vesicle located at the anterior end of the neural tube. To define regulatory sequences required for pigment cell-lineage-specific expression of HrTyr during embryogenesis, a series of mutations of the 5' upstream region of HrTyr were fused to the lacZ reporter gene and were microinjected into fertilized eggs. We found that the -152bp upstream of the translational start site is essential for expression in pigment cell precursors of tailbud-stage embryos. Further, additional positive and unique restriction elements were identified in the region up to -1.8kb. Surprisingly, in the -152bp minimal promoter or in other regions with regulatory activities, there are no E-box motifs or sequences correlating with other conserved elements regulating vertebrate tyrosinase promoters. The possibility that Pax proteins regulate HrTyr expression is also discussed.

In order to investigate the neural connection of planarian, it is imperative to produce an antibody that specifically stains axons. To identify axon-specific genes, we constructed a cDNA library from a single eye by using a single cell PCR method, in which visual neurons are major components, and sequenced one thousand independent clones. We succeeded in the identification of a planarian homologue of synaptotagmin, Djsyt, whose specific expression in neurons was confirmed by in situ hybridization. The antibody against DjSYT specifically stained axons although its mRNA is distributed in the cell bodies. By using anti-DjSYT, we succeeded in the visualization of neural connections in planarians by whole mount staining. The anti-DjSYT antibody will become a powerful tool to analyze the molecular mechanisms underlying neural network formation in planarian., (Copyright 1999 Academic Press.)

The tyrosinase family in vertebrates consists of three related melanogenic enzymes: tyrosinase, tyrosinase-related protein-1 (TRP-1), and TRP-2. These proteins control melanin production in pigment cells and play a crucial role in determining vertebrate coloration. We have isolated a gene from the ascidian Halocynthia roretzi which encodes a tyrosinase-related protein (HrTRP) with 45-49% identity with vertebrate TRP-1 and TRP-2. The expression of the HrTRP gene in pigment lineage a8.25 cells starts at the early-mid gastrula stage, which coincides with the stage when these cells are determined as pigment precursor cells; therefore, it provides the earliest pigment lineage-specific marker, which enables us to trace the complete cell lineage leading to two pigment cells in the larval brain. In addition, the expression pattern of the HrTRP gene appears to share similar characteristics with the mouse TRP-2 gene although structurally the HrTRP gene is more closely related to mammalian TRP-1 genes. Based on these observations and on results from molecular phylogenetic and hybridization analyses, we suggest that triplication of the tyrosinase family occurred during the early radiation of chordates. Initially, duplication of an ancestral tyrosinase gene produced a single TRP gene before the urochordate and cephalochordate-vertebrate divergence, and a subsequent duplication of the ancestral TRP gene in the vertebrate lineage gave rise to two TRP genes before the emergence of teleost fishes. Evolution of the melanin synthetic pathway and possible phylogenetic relationships among chordate pigment cells that accommodate the metabolic process are discussed. Dev Dyn 1999;215:225-237., (Copyright 1999 Wiley-Liss, Inc.)

Tadpole larvae of ascidians have two sensory pigment cells in the brain. One is the otolith cell that functions as a gravity receptor, the other pigment cell is part of a primitive photosensory structure termed the ocellus. These sensory cells, like vertebrate pigment cells, contain membrane-bounded melanin granules and are considered to reflect a crucial position in the evolutionary process of this cell type. To investigate the molecular changes accompanying the evolution of pigment cells, we have isolated from Halocynthia roretzi a gene encoding tyrosinase, a key enzyme in melanin biosynthesis. The cDNA has an open reading frame (ORF) of 596 amino acids, which is 36-39% identical in amino acid sequence to vertebrate tyrosinases. In addition, the sequence analysis of both cDNA and genomic clones reveals an unusual organization of the tyrosinase gene, an extraordinary 3' untranslated region of the transcripts with significant homology to the coding sequence, and a single short intron in the sequence encoding a cytoplasmic domain. Expression of the gene is detected first in two pigment precursor cells positioned in the neural plate of early neurulae, and later in two melanin-containing pigment cells within the brain of late tailbud embryos. Its expression pattern correlates well with the appearance of tyrosinase enzyme activity in the developing brain. These results provide the first description of pigment cell differentiation at the molecular level in the ascidian embryo, and also will contribute to a better understanding of the evolution of chordate pigment cells.

With the aim of elucidating the evolution of a hepadnavirus family, we constructed molecular phylogenetic trees for 27 strains of hepatitis B virus (HBV) using both the unweighted pair-grouping and neighbor-joining methods. All five gene regions, P, C, S, X, and preS, were used to construct the phylogenetic trees. Using the phylogenetic trees obtained, we classified these strains into five major groups in which the strains were closely related to each other. Our classification reinforced our previous view that genetic classification is not always compatible with conventional classification determined by serological subtypes. Moreover, constraints on the evolutionary process of HBV were analyzed for amino-acid-altering (nonsynonymous) and silent (synonymous) substitutions, because two-thirds of the open reading frame (ORF), P, contains alternating overlapping ORFs. In our unique analysis of this interesting gene structure of HBV, the most frequent synonymous substitutions were observed in the nonoverlapped parts of the P and C genes. On the other hand, the number of synonymous substitutions per nucleotide site for the S gene was quite low and appeared a strongly constrained evolution. Because the P gene overlaps the S gene in a different frame, the low rate of synonymous substitution for the S gene can be explained by the evolutionary constraints which are imposed on the overlapping gene region. In other words, synonymous substitutions in the S gene can cause amino acid changes in its overlapping region in a different frame. Thus, the evolution of HBV is constrained evolutionarily by the overlapping genes. We propose calling this mode of viral evolution "constrained evolution." The evolution of HBV represents a typical constrained evolution.

Two flavivirus-like viruses, GB virus-A (GBV-A) and GB virus-B (GBV-B), were recently identified in the GB hepatitis agent, and are distinct from the hepatitis A to E viruses. The putative helicase domain of GBV-A and GBV-B was found to have amino acid sequence homology with hepatitis C virus (HCV), and distantly, is also related to pestiviruses, flaviviruses, and plant viruses. A phylogenetic tree construction showed that GBVs and HCV are closely related, and they are clustered with pestiviruses, flaviviruses and plant viruses in that order.

Hepatitis B virus (HBV) surface antigen (HBsAg), which is encoded by the HBV S gene, is conventionally classified into 4 serological subtypes, adw, adr, ayw and ayr. To determine the relationship between the HBsAg seroreactivity and the nucleotide sequence diversity of the HBV S gene, the nucleotide sequences of S genes for HBV isolates reported so far were aligned with each other. The numbers of nucleotide substitutions were then estimated by the 6-parameter method, and a phylogenetic tree was constructed by the unweighted paired grouping method with arithmetic mean (UPGMA) and the neighboring-joining (NJ) method. The phylogenetic trees constructed showed that all isolates were grouped into 4 genotypes (gyw, gdw-1, gdw-2, and gdr). More importantly, the genotypes did not necessarily correspond to the conventional serotypes. In particular, serotype 'adw' can be any of genotypes gdw-1, gdw-2, or gdr. Thus, genotyping by S genes gives more accurate information about genetic variation of HBV.

With the aim of elucidating the evolutionary processes of the kringle and protease domains in serine proteases which are involved with the system of blood coagulation and fibrinolysis, we constructed phylogenetic trees for the kringle and protease domains, separately, by use of amino acid sequence data. The phylogenetic trees constructed clearly showed that the topologies were different between the kringle and protease domains. Because both domains are coded by single peptides of serine proteases, this strongly suggests that the kringle and protease domains must have undergone different evolutionary processes. Thus, these observations imply that serine proteases evolve in a way such that each domain is a unit of evolution, exemplifying a typical mode of domain evolution. A possible relationship between the domain evolution and the exon shuffling theory is also discussed from the viewpoint of gene evolution.

For pathogenic viruses such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), human influenza A virus, and human T-cell leukemia virus type I (HTLV-I), the evolutionary features were briefly reviewed with special reference to the rates of synonymous and nonsynonymous substitutions. In particular, these rates were discussed in connection with the neutral theory of molecular evolution. It was common to all the five pathogenic viruses that the rate of synonymous substitution was higher than that of nonsynonymous substitution particularly when the entire gene regions were compared between different isolates. This suggests that the viral proteins are quite conservative to functional and structural changes even though most of these viral genomes are evolving at a speed extraordinarily higher than their host genomes. Thus, this feature is consistent with the neutral theory. However, it is also pointed out that positive selection may be operating on some specific sites such as antigenic sites in order for the pathogenic viruses to escape from the host immune system.

The evolution of serine protease and its inhibitor are discussed with special reference to domain evolution. It is now known that most proteins are composed of more than one functional domain. Because serine proteases such as urokinase and plasminogen are made of various functional domains, these proteins are typical examples of the so-called mosaic proteins. When Kringle domains in serine proteases and a Kunitz-type protease inhibitor domain in the amyloid beta precursor protein in Alzheimer's disease patients were examined by the molecular evolutionary analysis, the phylogenetic trees constructed showed that these functional domains had undergone dynamic changes in the evolutionary process. In particular, these domains are evolutionarily movable. Thus, it is concluded that various functional domains evolved independently of each other and that they have been shuffled to create the existent mosaic proteins. This conclusion leads us to the reasonable speculation that those functional domains must have been minigenes possibly at the time of primordial life or the origin of life. We call these minigenes 'ancestral minigenes'. Every effort should be made to answer the question about the minimum set of ancestral minigenes that must have existed and must have been needed for maintaining life forms. The DNA sequence database is useful for making attempts to answer such difficult but significant questions.

Rad51, of Saccharomyces cerevisiae, is a homologue of recA of Escherichia coli and plays crucial roles in both mitotic and meiotic recombination and in repair of double-strand breaks of DNA. We have cloned genes from human, mouse and fission yeast that are homologous to rad51. The 339 amino acid proteins predicted for the two mammalian genes are almost identical and are highly homologous (83%) with the yeast proteins. The mouse gene is transcribed at a high level in thymus, spleen, testis and ovary and at a lower level in brain and other tissues. The rad51 homologues fail to complement the DNA repair defect of rad51 mutants of S. cerevisiae. The mouse gene is located in the F1 region of chromosome 2 and the human gene maps to chromosome 15.

The Kunitz-type protease inhibitor is one of the serine protease inhibitors. It is found in blood, saliva, and all tissues in mammals. Recently, a Kunitz-type sequence was found in the protein sequence of the amyloid beta precursor protein (beta APP). It is known that beta APP accumulates in the neuritic plaques and cerebrovascular deposits of patients with Alzheimer's disease. Collagen type VI in chicken also has an insertion of a Kunitz-type sequence. To elucidate the evolutionary origin of these insertion sequences, we constructed a phylogenetic tree by use of all the available sequences of Kunitz-type inhibitors. The tree shows that the ancestral gene of the Kunitz-type inhibitor appeared about 500 million years ago. Thereafter, this gene duplicated itself many times, and some of the duplicates were inserted into other protein-coding genes. During this process, the Kunitz-type sequence in the present beta APP gene diverged from its ancestral gene about 270 million years ago and was inserted into the gene soon after duplication. Although the function of the insertion sequences is unknown, our molecular evolutionary analysis shows that these insertion sequences in beta APP have an evolutionarily close relationship with the inter-alpha-trypsin inhibitor or trypstatin, which inhibits the activity of tryptase, a novel membrane-bound serine protease in human T4+ lymphocytes.

The 32P-labeled urokinase (uPA) bound to surface receptors of Detroit 562 cells was immunoprecipitated by anti-uPA antibody. Amino acid analysis showed that tyrosines and serines were the acceptors. Inhibition of protein kinases greatly reduced the 32P incorporation, suggesting that the respective cellular src gene product and protein kinase C were involved in the phosphorylations. Proteins purified on chromatographic columns contained two forms of uPA, a high (HMW) and a low (LMW) molecular weight. Tyrosine-phosphorylation occurs in the HMW and A-chain. Such modifications might modulate the extracellular activities of uPA.

Human apolipoprotein(a) has a great size heterogeneity and consists of 38 kringle domains in the amino terminal and a serine protease domain in the carboxyl terminal. All but one kringle of apolipoprotein(a) are homologous to the fourth kringle of plasminogen. However, the 38th kringle resembles the fifth kringle of plasminogen and its seems to have been deleted in simian species. The phylogenetic trees suggest that an ancestral apolipoprotein(a) may have started with a duplicate of a plasminogen type protein. It also implies that deletion of the three kringles in the amino terminus followed, and that one of the remaining two kringles was duplicated in both human and simian species and the other was processed by a deletion in simian species after species separation. Thus, the number of kringles in other mammals not yet studied may vary considerably from species to species.

Human immunodeficiency viruses (HIVs) show extensive genetic variation. This feature is the fundamental cause of pathogenicity of HIVs and thwarts efforts to develop effective vaccines. To understand the mutation mechanism of these viruses, we analysed nucleotide sequences of env and gag genes of the viruses by use of molecular evolutionary methods and estimated the direction and frequency of nucleotide substitutions. Results obtained showed that the frequency of changes between A and G was extremely high and the mutation pattern of HIVs was distinct from those of nuclear genes of their host cells. This distinction may be caused by the characteristics of the reverse transcription of HIVs. The mutation pattern obtained would be helpful to construct effective antiviral drugs.

The local activity of urokinase and its receptor associated with a cloned cell (C5) obtained from the cloning of Detroit 562 was investigated. The cellular binding sites, similar structure to adhesion plaques, were visualized by fluorescein labeled urokinase and the number was determined to be 300 per cell. The binding sites for radioiodinated urokinase were found to be 30 thousand per cell. Thus, about as many as 100 receptor molecules per site was estimated to be associated with the cellular membrane domains. Immunofluorescence studies demonstrated that the receptors were colocalized with a set of adhesion and cytoskeletal proteins such as vinculin, alpha-actinin and actin; localizing at the adhesion sites. These proteins soluble in 9 M urea were able to be reconstituted by dialyzing out the urea against low ionic buffer solution. It was demonstrated that vinculin and actin were co-associated. Since cell bound urokinase revealed fibrinolytic activity, it was suggested that the focal adhesions of the migrating cells would facilitate proteolytic action when cells move across the matrix architectures.