Comparative sequence analysis of the human and pufferfish Huntington's disease genes.

The Huntington's disease (HD) gene encodes a novel protein with as yet no known function. In order to identify the functionally important domains of this protein, we have cloned and sequenced the homologue of the HD gene in the pufferfish, Fugu rubripes. The Fugu HD gene spans only 23 kb of genomic DNA, compared to the 170 kb human gene, and yet all 67 exons are conserved. The first coding exon, the site of the disease-causing triplet repeat, is highly conserved. However, the glutamine repeat in Fugu consists of just four residues. We also show that gene order may be conserved over longer stretches of the two genomes. Our work describes a detailed example of sequence comparison between human and Fugu, and illustrates the power of the pufferfish genome as a model system in the analysis of human genes.

Small is beautiful: comparative genomics with the pufferfish (Fugu rubripes).

As the Human Genome Project advances, it is clear that the emphasis will switch from accumulation of data to their interpretation. Comparative genomics provides a powerful way in which to analyse sequence data. Indeed, there is already a long list of 'model' organisms, which allow comparative analyses in a variety of ways. The very small vertebrate genome of the pufferfish provides a simple and economical way of comparing sequence data from mammals and fish, representing a large evolutionary divergence and so permitting the identification of essential elements that are still present in both species. These elements include genes and the associated machinery that controls their expression; elements that, in many cases, have survived the test of time.

Statistical information characterization of conserved non-coding elements in vertebrates.

Recently, a set of highly conserved non-coding elements (CNEs) has been derived from a comparison between the genomes of the puffer fish, Takifugu or Fugu rubripes, and man. In order to facilitate the identification of these conserved elements in silico, we characterize them by a number of statistical features. We found a pronounced information pattern around CNE borders; although the CNEs themselves are AT rich and have high entropy (complexity), they are flanked by GC-rich regions of low entropy (complexity). We also identified the most abundant motifs within and around of CNEs, and identified those that group around their borders. Like in human promoter regions, the TBP, NF-Y and some other binding motifs are clustered around CNE boundaries, which may suggest a possible transcription regulatory function of CNEs.

Enzymatic and immunological evidence for two forms of carnosinase in the mouse.

Carnosinase (aminoacyl-L-histidine hydrolase, EC 3.4.13.3) hydrolyzes the dipeptide carnosine (beta-alanyl-L-histidine), which is thought to play a role in cerebral and skeletal muscular function and has been implicated as a neuroaffector in the olfactory bulb. Carnosinase activity is present in many tissues of the mouse including heart, liver and lung, but it is most active in kidney, uterus and nasal olfactory mucosa. Kinetic measurements with 1H-NMR spectroscopy indicate that the enzyme is stereospecific and can hydrolyze L-but not D-carnosine. Anserine is a poorer substrate, while homocarnosine is essentially a non-substrate. However, these two dipeptides are effective inhibitors of the hydrolysis of L-carnosine. Carnosinase activity is unaffected when assayed in 2H2O at 99% isotopic purity. From considerations of the effect of Mn2+ on (1) substrate concentration velocity curves; (2) thermostability, and (3) inhibitor behavior, tissues with carnosinase can be divided into two groups. Kidney, uterus and olfactory mucosa represent one group, while central nervous system, muscle, spleen, etc. represent the second. The validity of this classification is confirmed by immunological evidence. Antiserum prepared against carnosinase purified from kidney cross-reacts with and inhibits the activity of olfactory mucosa, kidney and uterus but not that from central nervous system, heart or liver.

The identification and characterization of microsatellites in the compact genome of the Japanese pufferfish, Fugu rubripes: perspectives in functional and comparative genomic analyses.

Fugu rubripes (Fugu) has one of the smallest recorded vertebrate genomes and is an economic tool for comparative DNA sequence analysis. Initial characterization of 128 kb of Fugu DNA attributed the compactness of this genome, in part, to a sparseness of repetitive DNA sequence compared with mammalian genomic sequences. This paper describes a new and comprehensive analysis in which 501 theoretically possible microsatellites with a repeat unit of one to six bases were used to query two orders of magnitude more Fugu DNA (i.e. 11.338 Mb). A total of 6042 microsatellites were identified and categorized. In decreasing order, the 20 most frequently occurring microsatellites are AC, A, C, AGG, AG, AGC, AAT, AAAT, ACAG, ACGC, ATCC, AAC, ATC, AGGG, AAAG, AAG, AAAC, AT, CCG and TTAGGG. The 20 most frequently occurring microsatellites represent 81.79% of all microsatellites identified. Our results indicate that one microsatellite occurs every 1.876 kb of DNA in Fugu, 11.55% of the microsatellites are detected in open reading frames that are predicted protein coding regions. With respect to the proportion of microsatellites present in open reading frames and the total abundance (bp) of all microsatellites, the genome of Fugu is similar to the genome of many other vertebrate species. Previous estimates performed indicate that approximately 1% of many vertebrate genomes are comprized of microsatellite sequences. However, many differences prevail in the abundance and frequency of the individual microsatellite classes. Many of the frequently occurring microsatellites in Fugu are known to code in other species for regions in proteins such as transcription factors, whilst others are associated with known functions, such as transcription factor binding sites and form part of promoter regions in DNA sequences of genes. Therefore, it is likely that such repeats in genomes have a role in the evolution of genes, regulation of gene expression and consequently the evolution of species.

Isolation, characterization and evolution of nine pufferfish (Fugu rubripes) actin genes.

The Japanese pufferfish Fugu rubripes (Fugu) has a small genome of about 400 Mb. Nine different actin genes have been isolated and sequenced from a genomic library constructed from this teleost. The six muscle-type actin genes include two alpha-skeletal actins, three alpha-cardiac actins and an alpha-anomalous (testis type) actin, and the three cytoplasmic actins include two beta-cytoplasmic actins and a beta-cytoplasmic (vascular type) actin. The two skeletal muscle actin genes have identical genomic organization, but differ by five amino acid residues. The three cardiac actin genes code for the same protein but differ in their nucleotide sequences and genomic organization. beta-Cytoplasmic actin1 differs by three amino acids from beta-cytoplasmic actin2. The alpha-anomalous (testis type) and beta-cytoplasmic (vascular type) actins are novel vertebrate actins. The amino acid sequence of alpha-anomalous (testis type) actin is the most divergent of all the known vertebrate actins and transcripts of this gene are abundant in the testis. The beta-cytoplasmic (vascular type) actin gene has eight introns, similar to mammalian smooth muscle actins, and is expressed in vascular tissues such as the gills, kidney and skin. Several known regulatory elements are found in the 5' flanking sequences and the first intron of various Fugu actin genes. The intron patterns of the various Fugu actins seem to be the result of loss of certain introns from a common ancestral gene.

Identification and analysis of two snail genes in the pufferfish (Fugu rubripes) and mapping of human SNA to 20q.

All members of the snail gene family are zinc-finger transcription factors expressed early in embryonic development and are involved in the formation of tissues such as mesoderm and presumptive neural crest. Here, we report the identification and structural organisation of two snail genes in the compact genome of the pufferfish Fugu rubripes, and examine the phylogenetic relationships between these and other members of the snail gene family. Both genes have a three exon, two intron structure similar to that previously reported for human SLUG. While human SLUG has been mapped to 8q (Cohen, M.E., Yin, M., Paznekas, W.A., Schertzer, M., Wood, S., Jabs, E.W., 1998. Human SLUG organisation, expression and chromosome map location on 8q. Genomics 51, 468-471), the human sna gene SNA, was previously unmapped. We have used sequence similarity to the Fugu genes to identify a human SNA EST and mapped this by radiation hybrid and physical mapping to the distal end of human 20q. This is likely to be the mapping location of the human sna gene (SNA).

Characterisation of two topoisomerase 1 genes in the pufferfish (Fugu rubripes).

Eukaryotic DNA topoisomerase I manipulates the higher order structures of DNA. Only one functional topoisomerase 1 (top1) gene has previously been identified in any individual eukaryotic species. Here we report the identification and characterisation of two top1 genes in the pufferfish, Fugu rubripes. This shows that the copy number of top1, like that of other topoisomerases, may vary between eukaryotes. Both Fugu genes have 21 exons; a gene structure similar to that of human TOP1. Despite this conservation of structure, and some non-coding elements, both genes are less than a tenth of the size of the human gene. Sequence and phylogenetic analyses have shown that this duplication is ancient and also affects other species in the fish lineage.

Characterization of three genes, AKAP84, BAW and WSB1, located 3' to the neurofibromatosis type 1 locus in Fugu rubripes.

Sequence analysis of cosmid clones was instrumental to identify three genes in the region flanking the Fugu rubripes NF1 gene in the 3' direction: the AKAP84 gene (A-kinase anchor protein 84), the WSB1 gene (WD-40-repeat protein with a SOCS box) and the BAW gene of yet unknown function located between the AKAP84 and the WSB1 genes. The human homologues of these genes are not located in the immediate vicinity of the NF1 gene at 17q11.2. Although synteny of the NF1, AKAP84, BAW and WSB1 genes is conserved between Fugu and human, the gene order is not conserved, and more than a simple inversion would have been necessary to explain the difference in gene order. The mammalian homologue of the Fugu BAW gene or protein has not yet been characterized. As deduced from the respective cDNAs, the Fugu AKAP84, WSB1 and BAW proteins vary concerning the overall degree of similarity to their mammalian counterparts. Whereas the overall similarity of AKAP84 between Fugu and mouse is low, three regions of known functional importance show considerable conservation. These are the N-terminal anchoring domain mediating the insertion of AKAP84 in the outer mitochondrial membrane, the binding site of the regulatory subunit (RI or RII) of protein kinase A, and the C-terminal domain present in the alternatively spliced isoform AKAP121 with an hnRNP K homology domain involved in RNA binding. A higher overall similarity of deduced protein sequences between Fugu and mouse was observed comparing the BAW gene products (74.1%) and the WSB1 proteins (77.2%).

Genomic characterization of the Neurofibromatosis Type 1 gene of Fugu rubripes.

The genomic structure of the Neurofibromatosis Type1 (NF1) gene of Fugu rubripes was investigated by sequence analysis of two overlapping cosmids. The Fugu NF1 gene spans 27 kb and is 13 times smaller than the human counterpart owing primarily to reduced intron size. The predicted amino acid sequence is highly related to that of human neurofibromin, exhibiting an overall similarity of 91.5%. Nearly all exons described for the human NF1 gene could be identified, except exon 12b and the alternatively spliced exons 9br and 48a. With the exception of the splice acceptor site in front of exon 16, all splice sites are in identical positions to those found in the human gene. Intron 1, which is 100-140 kb long in humans, spans 2575 bp in the Fugu NF1 gene. Another large intron of the human NF1 gene, intron 27b (45-50 kb), is 3942 bp of size in Fugu. Sequences related to the OMgp gene (Oligodendrocyte-Myelin-glycoprotein) or the EVI2A gene (ecotropic viral integration site), which are inserted into human NF1 intron 27b, were not detected in the corresponding Fugu intron. However, a single exon gene with similarity to the human EVI2B gene has been found on the reverse strand of Fugu intron 27b. This suggests that the human EVI2B gene and the Fugu gene in intron 27b have a common ancestor. We found the expression of this inserted gene in liver and kidney, but not in brain tissue of Fugu rubripes.

Cloning and sequencing of complement component C9 and its linkage to DOC-2 in the pufferfish Fugu rubripes.

The Japanese pufferfish Fugu rubripes has a 400 Mb genome with high gene density and minimal non-coding complexity, and is therefore an ideal vertebrate model for sequence comparison. The identification of regions of conserved synteny between Fugu and humans would greatly accelerate the mapping and ordering of genes. Fugu C9 was cloned and sequenced as a first step in an attempt to characterize the region in Fugu homologous to human chromosome 5p13. The 11 exons of the Fugu C9 gene share 33% identity with human C9 and span 2.9 kb of genomic DNA. By comparison, human C9 spans 90 kb, representing a 30-fold difference in size. We have also determined by cosmid sequence scanning that DOC-2, a tumour suppresser gene which also maps to human 5p13, lies 6-7 kb from C9 in a head-to-head or 5' to 5' orientation. These results demonstrate that the Fugu C9/DOC-2 locus is a region of conserved synteny. Sequence scanning of overlapping cosmids has identified two other genes, GAS-1 and FBP, both of which map to human chromosome 9q22, and lie adjacent to the Fugu C9/DOC-2 locus, indicating the boundary between two syntenic regions.

Characterization of the Fugu rubripes NLK and FN5 genes flanking the NF1 (Neurofibromatosis type 1) gene in the 5' direction and mapping of the human counterparts.

To complete the analysis of the Neurofibromatosis type 1 (NF1) gene region in Fugu rubripes, we characterized the upstream flanking region of the NF1 gene and identified the FN5 (flanking the Fugu NF1 gene in 5' direction) gene and the NLK (Nemo-like kinase) gene as its flanking genes. The FN5 gene spans 3807bp and encompasses four exons, three of which belong to the expanded 5' UTR. Only 11% of the FN5 transcript is protein-coding. The function of the FN5 protein spanning 59 amino acids is unknown. We also characterized the human and the mouse FN5 transcripts and found 85% and 83% similarity of deduced amino acid sequences compared with Fugu. Two copies of the human FN5 gene were identified, one on chromosome 17q21.3-q22 several megabases distal to the NF1 gene at 17q11.2. The second copy of the FN5 gene was mapped to 11q13.3-q23.3. In Fugu, the FN5 gene is flanked by the NLK gene, which spans 4513bp from the translation start to the stop codon and encompasses 11 exons. Comparing the deduced amino acid sequences, 82% overall similarity was observed between Fugu and mouse or human NLK and 67% similarity between the Fugu NLK and the highly related LIT-1 kinase of Caenorhabditis elegans, which has been shown, like the vertebrate counterpart, to be involved in the Wnt signalling pathway. We mapped the human NLK gene to 17q11.2 between markers D17S935 and D17S120, more than 1Mb proximal to the NF1 gene. The characterization of the 5' flanking region presented here, together with that of the 3' region, demonstrates the profound differences between Fugu and human considering the gene content within the region flanking the NF1 gene.

Isolation and characterisation of the retinoic acid receptor-alpha gene in the Japanese pufferfish, F. rubripes.

Nuclear hormone receptors (NRs) are ligand-inducible transcription factors that mediate critical functions in many species. The majority of novel NRs have hitherto been cloned from cDNA libraries by virtue of their homology to previously identified receptors. In this study, we validate a genomic DNA-based approach to isolating NRs by cloning the retinoic acid receptor-alpha (RARalpha) gene from the genome of the Japanese pufferfish, Fugu rubripes. The fRARalpha gene is more compact than its human and murine counterparts and demonstrates a highly conserved genomic organisation and amino acid sequence, generating two isoforms (fRARalpha1 and fRARalpha2) with divergent aminoterminal domains. In addition, a conserved regulatory element containing a retinoic acid response element was identified upstream of the fRARalpha2-specific exon, implying that retinoid induction of this isoform is evolutionarily conserved and critical to its function in vivo. We propose two uses for the Fugu genome in the study of NRs: the isolation of novel NRs that exhibit restricted spatio-temporal expression from genomic DNA and the identification of evolutionarily conserved promoter or intragenic regulatory DNA elements.

Sequence scanning chicken cosmids: a methodology for genome screening.

The chicken genome is relatively poorly studied at the molecular level. The karyotype 2n=78 is divided into three main chromosomal sub-groups: the macrochromosomes (six pairs), the intermediate microchromosomes (four pairs) and the microchromosomes (29 pairs). Whilst the microchromosome group comprise only 25% of the DNA, increasing evidence is proving that this is disproportionate to their gene content. This paper demonstrates the utility of cosmid sequence scanning as a potential method for analysing the chicken genome, providing an economical method for the production of a molecular map. The GC content, gene density and repeat distribution are analysed relative to chromosomal origin. Results indicate that gene density is higher on the microchromosomes. During the scanning process an example of conserved linkage between chicken and human (12q34.2) has been demonstrated.

The neural cell adhesion molecule L1: genomic organisation and differential splicing is conserved between man and the pufferfish Fugu.

The human gene for the neural cell adhesion molecule L1 is located on Xq28 between the ALD and MeCP2 loci. Mutations in the L1 gene are associated with four related neurological disorders, X-linked hydrocephalus, spastic paraplegia (SPG1), MASA syndrome, and X-linked corpus callosum agenesis. The clinical relevance of L1 has led us to sequence the L1 gene in human and to investigate its conservation in the vertebrate model genome of the pufferfish, Fugu rubripes (Fugu), a species with a compact genome of around 40Mb. For this purpose we have sequenced a human and a Fugu cosmid clone containing the corresponding L1 genes. For comparison, we have also amplified and sequenced the complete Fugu L1 cDNA. We find that the genomic structure of L1 is conserved. The human and Fugu L1 gene both have 28 exons of nearly identical size. Differential splicing of exons 2 and 27 is conserved over 430 million years, the evolutionary time span between the teleost Fugu and the human L1 gene. In contrast to previously published Fugu genes, many introns are larger in the Fugu L1 gene, making it slightly larger in size despite the compact nature of the Fugu genome. Homology at the amino acid and the nucleotide level with 40% and 51%, respectively, is lower than that of any previously reported Fugu gene. At the level of protein structure, both human and Fugu L1 molecules are composed of six immunoglobulin (Ig)-like domains and five fibronectin (Fn) type III domains, followed by a transmembrane domain and a short cytoplasmic domain. Only the transmembrane and the cytoplasmic domains are significantly conserved in Fugu, supporting their proposed function in intracellular signalling and interaction with cytoskeletal elements in the process of neurite outgrowth and fascicle formation. Our results show that the cytoplasmic domain can be further subdivided into a conserved and a variable region, which may correspond to different functions. Most pathological missense mutations in human L1 affect conserved residues. Fifteen out of 22 reported missense mutations alter amino acids that are identical in both species.

Genomic structure and expression of parathyroid hormone-related protein gene (PTHrP) in a teleost, Fugu rubripes.

In this study we describe the isolation and characterisation of the parathyroid hormone-related protein (PTHrP) gene from the teleost Fugu rubripes. The gene has a relatively simple structure, compared with tetrapod PTHrP genes, composed of three exons and two introns, encompassing 2.25kb of genomic DNA. The gene encodes a protein of 163 amino acids, with a putative signal peptide of 37 amino acids and a mature peptide of 126 amino acids. The overall homology with known tetrapod PTHrP proteins is low (36%), with a novel sequence inserted between positions 38 and 65, the absence of the conserved pentapeptide (TRSAW) and shortened C-terminal domain. The N-terminus shows greater conservation (62%), suggesting that it may have a hypercalcaemic function similar to that of tetrapod PTHrP. In situ localisation and RT-PCR have demonstrated the presence of PTHrP in a wide range of tissues with varying levels of expression. Sequence scanning of overlapping cosmids has identified three additional genes, TMPO, LDHB and KCNA1, which map to human chromosome 12, with the latter two mapping to 12p12-11.2. PTHrP in human also maps to this chromosome 12 sub-region, thus demonstrating conservation of synteny between human and Fugu.

Three receptor genes for plasminogen related growth factors in the genome of the puffer fish Fugu rubripes.

Plasminogen related growth factors (PRGFs) and their receptors play major roles in embryogenesis, tissue regeneration and neoplasia. In order to investigate the complexity and evolution of the PRGF receptor family we have cloned and sequenced three receptors for PRGFs in the teleost fish Fugu rubripes, a model vertebrate with a compact genome. One of the receptor genes isolated encodes the orthologue of mammalian MET, whilst the other two may represent Fugu rubripes orthologues of RON and SEA. This is the first time three PRGF receptors have been identified in a single species.

Calcitonin: characterisation and expression in a teleost fish, Fugu rubripes.

The present report describes the structure and expression of the calcitonin gene in Fugu rubripes. It is composed of 4 exons and 3 introns. Splicing of exons 1, 2 and 3 generates the calcitonin pre-proprotein, while splicing of exons 1, 2 and 4 generates calcitonin gene-related protein (CGRP). Exons 1 and 2 encoding the signal sequence and the N-terminal peptide are common in both the gene products and this gene organisation has been conserved in human, rat, chicken and salmon. The gene environment around calcitonin in Fugu has been poorly conserved when compared with human, apart from a small gene cluster. The calcitonin gene in Fugu has a widespread tissue distribution but it is most highly expressed in the brain. The abundance of gene expression in the ultimobranchial gland and the pituitary indicates that these are important sites of production and that the peptide is probably secreted into the circulation and/or acts as a paracrine or autocrine controlling factor. Whilst the function of calcitonin in fish is still largely unknown, the distribution described here suggests that one of the potential functions may be as a neuropeptide.

Duplicated receptors for VIP and PACAP (VPAC1R and PAC1R) in a teleost fish, Fugu rubripes.

Two principal groups of receptors orthologous with human PAC1R and VPAC1R and were identified and characterised at the genomic level in the teleost fish Fugu rubripes. An additional group orthologous with VPAC2R was also identified and partially characterised. In Fugu, gene duplication of each of the PAC1Rs, VPAC1Rs and VPAC2Rs appears to have occurred. The topology of the tree surrounding the Fugu duplications and other isolated piscine sequences indicates that the duplication events for these six genes clearly preceded the speciation event leading to the Cypriniformes and Tetraodontiformes and is probably teleost-specific. Overall, the combined pattern of gene expression for each pair of duplicated genes mirrored the expression in other vertebrates. However, within each pair of duplicates further specialisation had occurred, with each demonstrating differential tissue distribution profiles suggesting they that may be responsible for the divergent action of the ligands, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP). The Fugu VPAC1R gene regions showed conserved synteny with human chromosome 3p21.3 and also C. elegans chromosome X, indicating that the putative ancestral human chromosome 3 region may be equivalent to chromosome X in Caenorhabditis elegans.

Use of the Japanese pufferfish (Fugu rubripes) in comparative genomics.

With the draft sequence of the human genome available and an increasing number of organisms being sequenced, attention is becoming focused on sequence interpretation and functional analysis. Comparative genomics will play an important role in evaluating these data. At the molecular level, roles for uncharacterized proteins can be hypothesized by identifying conserved protein domains and putative noncoding regulatory elements can be defined from direct sequence comparisons of evolutionarily distant organisms. At a higher level, questions, such as the importance of gene order positioning, conservation of linkage, and genome evolution, can begin to be answered by collecting map data from different organisms. This minireview, centering on Fugu regions sharing synteny with human chromosomes 11p, 20q, and 6p21.3, details some of the ways in which the Japanese pufferfish can contribute to the study of comparative genomics and evaluation of sequence data from the genome programs.