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ILAR Journal V39(2/3) 1998
Comparative Gene Mapping
Greg Elgar and Melody Clark
| Greg Elgar, Ph.D., is Group Leader, and Melody Clark, Ph.D., is Senior Scientist with the United Kingdom Human Genome Mapping Project Resource Centre, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom. |
INTRODUCTION
The earliest true vertebrates are represented by fish. The largest fish class, the teleosts or bony fish, contain nearly 20,000 known species. Although this class comprises only about half of all the known vertebrate species, they are probably the least well studied at the molecular level among vertebrate phyla. Until studies of the mid-1990s on the zebrafish (Danio rerio) and the pufferfish (Fugu rubripes) (2 very different teleosts), only a handful of fish genes have been listed in databases. However, because of the evolutionary position of fish, many important aspects of molecular evolution may be addressed by their study.
The genomic organization of fish is very different from that of mammals. Fish chromosomes on the whole are much smaller than those of mammals. The way in which the chromosomes are constructed has a different organization. For example, fish chromosomes do not show Giemsa banding, which has been attributed to the fact that they have a less well-defined isochore structure, that is, they do not posses guanine and cytosine-poor and guanine and cytosine-rich regions (Medrano and others 1988). They also have a higher frequency of the dinucleotide CpG (Elgar 1996), the suppression of which is associated with the 5 prime ends of genes in mammals. This article and others in this Journal issue address whether such differences in the organization of other species' genes along respective chromosomes bears any resemblance to mammalian gene orders.
Pufferfish are members of the Tetraodontiformes, denoting 4 teeth. The approximately 200 to 300 species in the order are found in freshwater, brackish, and marine environments. F. rubripes (also called ToraFugu or TakiFugu) (Figure 1) are marine fish found in the temperate coastal waters of Japan. Because of their size and growth rate (potentially 60 cm long and weighing more than 2 kg), they are farmed commercially and eaten as a delicacy. Since they are known to be aggressive, however, they require a large marine tank. Because they do not mature sexually for 2 to 3 yr, they are difficult and unsuitable for breeding. F. rubripes, like many of the other 23 members of the genus Takifugu, have a diploid chromosome number of 2n=44 (Miyaki and others 1995).
REASONS FOR MAPPING THIS SPECIES
Although the lack of genetics (that is, classical linkage studies through interspecific backcross analysis) is seen by many as a limitation of the utility of the pufferfish (Fugu) as a model, it has never been the intention to promote Fugu in this way. Rather, the aim has been to study the physical genome, concentrating purely at the DNA and RNA levels, with particular emphasis on comparative gene structure and organization studies.
The pufferfish (Fugu) haploid genome size of approximately 400 Mb has been confirmed by a variety of methods. In spite of its small size, the genome resembles much larger mammalian genomes in gene number, gene structure, and homology (Brenner and others 1993). Due to the large (400 million yr) evolutionary gap between teleost fish and mammals, the only elements that appear to be conserved are the essential ones. This property of the Fugu genome has been used to identify regulatory elements from mammals by comparative sequence analysis (Aparicio and others 1995: Marshall and others 1994; Popperl and others 1995). Thus, the Fugu genome provides a template for studying the more "cluttered" genomes of other vertebrates (Elgar and others 1996).
Recent evidence from work focused on vertebrate Hox clusters has suggested that at least some fish may have undergone a round of genome duplication after the divergence of tetrapods from the lineage (possibly 200 to 300 million yr ago). Mammals have 4 Hox clusters (a to d), whereas the zebrafish has at least 7 (Vogel 1998), including duplicates of clusters a, b, and c. Despite extensive analysis, the Fugu genome appears to have only 4 clusters (Aparicio and others 1997), although it is possible that the 4th is a duplicate Hoxa cluster and that the d cluster has either been lost or not yet identified. It is clear, however, that Fugu has a coding content similar to that of mammals (Brenner and others 1993) and that gene families appear to have a similar gene complement to those of mammals (Macrae and Brenner 1995). These characteristics do not preclude Fugu from having undergone an additional, fish-specific round of genome duplication but suggest that if it did, then most of this duplication was probably lost very rapidly since there simply is not adequate space in the genome for twice as many genes as mammals have.
The objective of this article is to describe the endeavors of pufferfish genomics at the time of this writing. Because of the lack of genetic data, there is no genetic map on which to position sequences. Thus, an alternative and complementary approach has been taken that generates high-resolution data across small regions via cosmid sequence scanning. This is in contrast to the general trend, underpinned by genetic maps, which generates low-resolution data across the whole genome.
CURRENT MAP STATUS
As a result of the Fugu high-resolution data-gathering approach described above, more than 1000 cosmids have been sample sequenced, and analysis as of July 1998 has determined a number of conserved gene linkages with human chromosomes. We present a series of conserved linkages between Fugu and humans in which 2 or more genes map to the same Fugu cosmid or region and to a single human chromosome (Table 1). Some of these linkages are published and are acknowledged in the legend. Table 1 does not present an exhaustive listing of linkages in Fugu since analysis inevitably lags far behind sequence generation.
Of the several problems associated with this type of study, the most critical is the attempt to establish that sequence from the Fugu genome that will represent the true orthologue of a gene (homologous genes from different species that are descended from the same gene in the nearest common ancestor) and not simply an homologue (a similar member of a gene family). In many cases, conserved linkage with another gene in 2 different genomes is evidence that 2 genes are orthologues. For instance, if genes A, B, and C are found together in species X, and genes a, b, and c are found together in species x (where A is orthologous to a and C is orthologous to c), then one can reasonably assume (given good similarity) that B is orthologous to b, even if B/b are members of large gene families. An additional problem is the lack of high definition mapping data (that is, the exact order of genes on 2 chromosomal segments) in other species up to thc time of this issue's publication. When mapped, genes tend to be arranged according to genetic markers that are spread quite widely. It is not possible to identify an absolute gene order until a region has been fully characterized. This ultimately requires sequencing of the region, which is now under way for the human genome and across regions of other, now accurate comparative maps.
Increasingly, numerous evolutionary breakpoints are being identified in Fugu. In Table 2, Fugu cosmids that contain genes from 2 distinct regions of the human genome are listed. At the time of this writing, when no additional investigations have been carried out on these particular cosmids, the limits of the breakpoints have not been defined. Thus, it is not yet possible to determine the size of the evolutionarily conserved segments in Fugu.
APPROACHES USED TO DEVELOP THE MAP
To generate a reliable set of gene data, it is necessary to undertake a large-scale, genome-wide assessment. Such an assessment has been carried out at the sequence level in the Fugu genome by sequence scanning techniques. Due to the high degree of similarity between Fugu and mammalian genes, particularly at the nondegenerate amino acid level (DNA sequence similarities tend to be statistically less significant because there are only 4 bases and because of codon wobble), and because of the high gene density, sequence scanning of genomic clones is a relatively easy and efficient way to identify genes in Fugu. To put this into perspective, compare the human genome (approximately 60,000 to 70,000 genes with 1 gene every 40 to 50 kb [1 gene per cosmid clone]) with the Fugu genome (a similar number of genes with 1 gene every 6 kb [6 or 7 genes per cosmid clone]). Thus, the sequence scanning of a cosmid clone is much more profitable in terms of Fugu data acquisition. It is possible to identify many genes and to acquire close-range physical linkage data that are available immediately, even though the gene order may not be accurate. Genes with some form of significant database homology to known genes from other species are the easiest to identify. Although not all genes are identifiable, the increasing amount of data in the databases is leading to the identification of more genes and their subsequent confirmation by comparative computer analysis.
The concept of sequence scanning is relatively new for 3 reasons:
- Sequence scanning has only limited use when used on very large genomes (such as human and mouse). The average length of a gene coding sequence in the databases is approximately 1.2 kb. Thus, with 60,000 to 70,000 genes in the human genome, only about 2.5 to 3% of the human genome comprises coding sequences. Sequence scanning a cosmid would yield, on average, only I gene. Sequence scanning larger clones would require a very high level of coverage to sequence an exon.
- Sequence scanning relies on database searches to define hits on genes. Until the mid-1990s, insufficient data in the databases precluded such an approach.
- Sequencing has been very costly. However, the current availability of more robust sequencing enzymes has eliminated the need to sequence from ultra pure templates, and the cost of sequencing has decreased accordingly.
Sequence scanning is an extremely economical and efficient way to assay the contents of a genome, particularly in gene-dense genomes such as that of Fugu. As a resource, its value greatly and continually increases because other sequence data entered independently into a database may shed some light (by homology) on already existing data. In addition, sequence scanning data do not have to be extremely accurate. No complex sequence assembly is necessary, it is not highly degenerate, and the databases are extremely tolerant of mistakes in sequencing, still allowing the identification of significant homologies.
A well-characterized, gridded Fugu cosmid library is publicly available and provides a common reference point for all clones isolated (http://hgmp.mrc.ac.uk/). Cosmids are sequence scanned by generating random shotgun libraries (by sonication) and sequencing approximately 50 clones from each cosmid library. This methodology provides single-pass sequence data for about 50% of the cosmid insert, in turn allowing identification of all but the smallest genes on the cosmid. For example, sequence scanning would be expected to identify 3 of 6 exons, assuming that homology (particularly at the amino acid level) is sufficient. Sequences are searched using the Basic Local Alignment Search Tool ("BLAST") (Altschul and others 1997) periodically against the SWISSPROT (Bairoch and Apweiler 1997), TREMBL (Bairoch and Apweiler 1997), and EMBL (Rodriguez-Tomi and others 1996) databases. All Fugu sequences in the Fugu database are being submitted to EMBL as GSS clones and all data from the project are publicly available on the World Wide Web (Web1).
Because data are stored on the Web page by cosmid clone and because most cosmids appear to contain more than 1 gene, it is possible to assign close physical linkage to genes in the Fugu genome and then to examine the relationship between these genes in other organisms. Finally, it is possible to assess potential regions of conserved synteny between Fugu and other genomes and to identify areas for more intensive study.
SCIENTIFIC CONTRIBUTIONS OF THE MAP
The Fugu cosmid library was first made available to the scientific community through the Fugu Landmark Mapping Project in 1996. Since then, interest in Fugu has expanded with the generation of numerous collaborative projects. At the time of this writing, Fugu projects cover a wide spectrum of studies that include the following:
- gene characterization and discovery (Baxendale and others 1995; Elgar and others 1995; Lira and others 1997; Mason and others 1995; Sandford and others 1997; Timon and others 1998; Yeo and others 1997;Verma-Kurvari and Johnson 1997; Venkatesh and Brenner 1997);
- gene mapping (Aparicio and others 1997; Elgar and others 1997; Gilley and others 1997; Sandford and others 1996a); synteny/linkage (How and others 1996; Lim and Brenner 1997; Sandford and others 1996b; Schofield and others 1997; Trower and others 1996);
- evolution (Aparicio and others 1997; Brenner and Corrochano 1996; Lim and Brenner 1995; McNaughton and others 1997), repeats (Edwards and others 1998);
- transposons (K. Miller, Imperial College, London, personal communication, 1988);
- transgenics (Cecconi and others 1995; Sathasivam and others 1997; Venkatesh and others 1997);
- gene regulation (Aparicio and others 1995; Crosio and others 1996; Marshall and others 1994);
- gene families (Koh and others 1997; Macrae and Brenner 1995; Sarwal and others 1996; Venkatesh and others 1996; Yamaguchi and Brenner 1997; Yamaguchi and others 1996); and
- splicing (Cecconi and others 1996).
ANTICIPATED FUTURE CONTRIBUTIONS OF THE MAP
Sample sequencing produces an overall picture of gene content and organization in Fugu. However, limitations include the lack of absolute gene order and the ability to correlate the data directly to traditional maps as well as the fact that genes are present only in fragments. We expect sequence scanning of the genome to continue; however, the technique will be driven largely by the requirements of collaborators who seek to identify particular regions for more intensive study rather than by in-house projects.
To extend some of the observations and speculations related to the phenomenon of conserved gene order between mammals and Fugu, some cosmid contigs are being built and sequence scanned, usually in collaboration with groups working on the equivalent human gene region. The ultimate objective is to progress to whole cosmid sequencing and high-level resolution comparative mapping.
Whole cosmid sequencing projects in Fugu have already shown that novel genes can be identified in Fugu syntenic regions (Trower and others 1996). The number of known mammalian genes is small compared with the total number present; hence, Fugu sequencing will inevitably move toward gene discovery, hypothetical genes analysis, and functional analysis. Genomic comparisons across large regions may allow the identification of conserved elements involved in the regulation of gene expression and any other sequences that are critically conserved across 400 million yr of evolution.
USES OF THE MAP AND ACCESSIBILITY
All data from the Fugu genome project are available on our Web site (http://fugu.hgmp.mrc.ac.uk/), which consists of a series of interlinked pages. These pages are designed to allow easy public access to meaningful data as soon as they become available. There may be only a few hours' delay between a sequence being loaded onto an automated sequencer and its appearance on the Web. Users may enter the database by a search of DNA, amino acid sequences, or keywords from a Basic Local Alignment Search Tool output; by requesting information on a specific cosmid or cosmid clone; or by viewing the overall statistics of the project. Other available features on the Web pages include general information about the fish and a complete, up-to-date set of project protocols. A flow chart overview of the Web site links appears in Figure 2.
Users may also download the entire database of sequences as a fiat file from the Web, although the amount of data makes this a major task. At the time of this writing, all Fugu sequences are transferred into the GSS section of the EMBL Data Library, with limited annotation (in collaboration with the European Bio-informatics Institute at Hinxton, Cambridge, United Kingdom) so that they may be searched in the same way as the rest of the public databases.
The easiest way for biologists to access data of particular interest is to carry out a keyword search. All cosmid clones subjected to database searches are sorted into "significant" hits using a program called maximal segment pair ("MSP") crunch, which was developed by Eric Sonnhammer in the Sanger Centre at Hinxton. The keyword search scans the MSP crunch output and then lists any hits by cosmid clone. Ways to access the relevant information produced by the search are shown in Figure 3.
It is important to carefully evaluate alignments before assuming that a particular gene has been found. Many of the EMBL (DNA) similarities are due to repeat sequences (such as microsatellites) from within the genomic sequence rather than being gene specific. Generally, amino acid similarities (from SWISSPROT and TREMBL searches) are more significant and are easier to interpret than those between DNA. This difference is partly due to the decreased likelihood of finding identical residues in amino acid sequences (20 amino acids versus 4 bases) in the same position resulting in less noise and partly because of the DNA degeneracy of the genetic code, which allows DNA sequence of only 33% identity code for identical amino acids (such as both AGA and CGG as codes for arginine). It is also important to be careful when searching for genes that are members of large gene families because short isolated sequence fragments are not always sufficient to allow differentiation between similar genes. Relative mapping data from another identifiable gene on the same cosmid may indicate the origin of the gene family member.
CONCLUSION
The sequence scanning approach has been successfully applied in Fugu to provide an overview of the genome. More intensive study in particular regions using whole cosmid sequencing will not only produce data on absolute gene order and conservation of gene structure but also will help in identifying regulatory elements. An increasing number of genes are being sequenced in both zebrafish and other fish species (such as carp and medaka), which will produce meaningful fish maps. In addition, with the emergence of zebra fish genetics, advantages of Fugu genomics can be effectively combined with zebra fish genetics in gene finding, sequencing, and functional characterization.
1Abbreviations used in this paper: EMBL, European Molecular Biology Laboratory; Web, World Wide Web.
ACKNOWLEDGMENTS
The Fugu Landmark Mapping Project is funded by the Medical Research Council. We particularly thank Stephen Meek, Sarah Smith, and Sarah Warner, who, with the authors, generated most of the data; and Gary Williams, Yagnesh Umrania, Yvonne Edwards, and Martin Bishop of the UK Human Genome Mapping Project Resource Centre Computing Department, without whom the construction of the Web pages and the analysis would not have been possible.
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TABLE 1 Physicially linked genes in Fugua
| Physically linked genes in Fugu | Distance apart (kb) | Human chromosome assignment |
| CNR1 GABRR1 | Both on 1 cosmid (1) | 6q14-q15 6q14-q21 |
| TH NAP2 IGFII | Within 10 kb of each other (2) | 11p15.5 11p 11p15.5 |
| PAH IGFI | Within 20 kb of each other (3) | 12q22 12q22-q24 |
| TSC2 ADPKDI | Within 1 kb of each other (4) | 16p13.3 16p13.3 |
| WNT1 ERBB3 ARF3 WNT10B | All on 1 cosmid (5) | 12q13 12q13 12q13 12q13 |
| FOS S31iii125 S20i15 7SL RNA gene ATF3 fos-like gene DLST | All on 1 cosmid (6) | 14q24.3 14q24.3 14q24.3 Unassigned Unassigned Unassigned 14q24. |
| HOXB cluster (9 genes) | 90 kb (7) | 17q21-22 |
| HOXC cluster (9 genes) | 66 kb (8) | 12q12-q13 |
| Immunogobulin cluster VH genes (at least 6 genes) | All on 1 cosmid (9) | 14q32.3 |
| SURF2 SURF4 ASS DNM1 GOLGA2 | All on 1 cosmid (10) | 9q34 9q34 9q34 9q34 9q32-q34.1 |
| SRD5A1 ADCY2 | 152B12 | 5p15 5p15.2-15.1 |
| WT1 RCN1 PAX6 | All on 1 cosmid (11) | 11p13 11p13 11p13 |
| MMP2 SLC6A2 | 011A17 | 16q12.2 16q12.2 |
| NTRKR3 RGS2 | 026G17 | 1q21-23 1q31 |
| TOP1 PLCG1 KIAA0181 | All on 1 cosmid (12) | 20q12-q13 20q12-q13 20q12-q13 |
| C4A CSNK2B CYP21 AIF1 | All on 1 cosmid (13) | 6p21.3 6p21.3 6p21.3 6p21.3 |
| C8A C8B | 126C21 | 1p32 1p32 |
| MTF1 IT5-P | 017D12 | 1p32 1p32 |
| CPS1 MAP2 MYL1 | All on 1 cosmid (14) | 2q135 2q34 2q33-q34 |
| PDGFRB CSF1R | 2.2 kb apart (15) | 5q33 5q33 |
TABLE 2 Evolutionary breakpoints in Fugua
| Physically linked genes in Fugu | Distance apart (kb) | Human chromosome assignment |
| G6PD | All in 1 60-kb contig (1) | Xq28 |
| GPD1 ADCY6 CACNB3 | Breakpoint | 12 12q13-q13 12q3 |
| C9 DOC2 | All on 1 cosmid (2) | 5p13 5013 |
| GAS1 FBP1 | Breakpoint | 9q21.3-q22.1 9q22 |
| MSH2 PIGF MTA1 | 042H13 | 2p16-p15/2p22-p2 2p21-16 unassigned |
| GCH1 | Breakpoint | 14q22 |
FIGURE 1 The Japanese pufferfish Fugu rubripes. This species, which can grow to more than 60 cm long, is farmed as a delicacy in Japan. The liver and gonads, however, contain a powerful neurotoxin, tetrodotoxin, which each year claims the lives of many gourmets who have consumed poorly prepared fish.
FIGURE 2 Links between Fugu Web pages.
FIGURE 3 Example of Fugu Web page data from a keyword search.
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