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Analysis of 148 kb of genomic DNA around the wnt1 locus of Fugu rubripes.

ABSTRACT: The analysis of the sequence of approximately 150 kb of a genomic region corresponding to the wnt1 gene of the Japanese pufferfish Fugu rubripes confirms the compact structure of the genome. Fifteen genes were found in this region, and 26.6% of the analyzed sequence is coding sequence. With an average intergenic distance of <5 kb, this gene density is comparable to that of Caenorhabditis elegans. The compactness of this region corresponds to the reduction of the overall size of the genome, consistent with the conclusion that the gene number in Fugu and human genomes is approximately the same. Eight of the genes have been mapped in the human genome and all of them are found in the chromosomal band 12q13, indicating a high degree of synteny in both species, Fugu and human. Comparative sequence analysis allows us to identify potential regulatory elements for wnt1 and ARF3, which are common to fish and mammals.

SUBMITTER: Gellner K

PROVIDER: S-EPMC310722 | biostudies-literature | 1999 Mar

REPOSITORIES: biostudies-literature

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Analysis of 148 kb of genomic DNA around the wnt1 locus of Fugu rubripes.

Gellner K K Brenner S S

Genome research 19990301 3

The analysis of the sequence of approximately 150 kb of a genomic region corresponding to the wnt1 gene of the Japanese pufferfish Fugu rubripes confirms the compact structure of the genome. Fifteen genes were found in this region, and 26.6% of the analyzed sequence is coding sequence. With an average intergenic distance of <5 kb, this gene density is comparable to that of Caenorhabditis elegans. The compactness of this region corresponds to the reduction of the overall size of the genome, consi ...[more]

PMID: 10077531

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Project description:BackgroundMembers of the C-type lectin domain (CTLD) superfamily are metazoan proteins functionally important in glycoprotein metabolism, mechanisms of multicellular integration and immunity. Three genome-level studies on human, C. elegans and D. melanogaster reported previously demonstrated almost complete divergence among invertebrate and mammalian families of CTLD-containing proteins (CTLDcps).ResultsWe have performed an analysis of CTLD family composition in Fugu rubripes using the draft genome sequence. The results show that all but two groups of CTLDcps identified in mammals are also found in fish, and that most of the groups have the same members as in mammals. We failed to detect representatives for CTLD groups V (NK cell receptors) and VII (lithostathine), while the DC-SIGN subgroup of group II is overrepresented in Fugu. Several new CTLD-containing genes, highly conserved between Fugu and human, were discovered using the Fugu genome sequence as a reference, including a CSPG family member and an SCP-domain-containing soluble protein. A distinct group of soluble dual-CTLD proteins has been identified, which may be the first reported CTLDcp group shared by invertebrates and vertebrates. We show that CTLDcp-encoding genes are selectively duplicated in Fugu, in a manner that suggests an ancient large-scale duplication event. We have verified 32 gene structures and predicted 63 new ones, and make our annotations available through a distributed annotation system (DAS) server http://anz.anu.edu.au:8080/Fugu_rubripes/ and their sequences as additional files with this paper.ConclusionsThe vertebrate CTLDcp family was essentially formed early in vertebrate evolution and is completely different from the invertebrate families. Comparison of fish and mammalian genomes revealed three groups of CTLDcps and several new members of the known groups, which are highly conserved between fish and mammals, but were not identified in the study using only mammalian genomes. Despite limitations of the draft sequence, the Fugu rubripes genome is a powerful instrument for gene discovery and vertebrate evolutionary analysis. The composition of the CTLDcp superfamily in fish and mammals suggests that large-scale duplication events played an important role in the evolution of vertebrates.

Project description:BACKGROUND: In fish breeding, it is essential to discover and generate fish exhibiting an effective phenotype for the aquaculture industry, but screening for natural mutants by only depending on natural spontaneous mutations is limited. Presently, reverse genetics has become an important tool to generate mutants, which exhibit the phenotype caused by inactivation of a gene. TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetics strategy that combines random chemical mutagenesis with high-throughput discovery technologies for screening the induced mutations in target genes. Although the chemical mutagenesis has been used widely in a variety of model species and also genetic breeding of microorganisms and crops, the application of the mutagenesis in fish breeding has been only rarely reported. RESULTS: In this study, we developed the TILLING method in fugu with ENU mutagenesis and high-resolution melting (HRM) analysis to detect base pair changes in target sequences. Fugu males were treated 3 times at weekly intervals with various ENU concentrations, and then the collected sperm after the treatment was used to fertilize normal female for generating the mutagenized population (F1). The fertilization and the hatching ratios were similar to those of the control and did not reveal a dose dependency of ENU. Genomic DNA from the harvested F1 offspring was used for the HRM analysis. To obtain a fish exhibiting a useful phenotype (e.g. high meat production and rapid growth), fugu myostatin (Mstn) gene was examined as a target gene, because it has been clarified that the mstn deficient medaka exhibited double-muscle phenotype in common with MSTN knockout mice and bovine MSTN mutant. As a result, ten types of ENU-induced mutations were identified including a nonsense mutation in the investigated region with HRM analysis. In addition, the average mutation frequency in fugu Mstn gene was 1 mutant per 297 kb, which is similar to values calculated for zebrafish and medaka TILLING libraries. CONCLUSIONS: These results demonstrate that the TILLING method in fugu was established. We anticipate that this TILLING approach can be used to generate a wide range of mutant alleles, and be applicable to many farmed fish that can be chemically mutagenized.

Dataset Information

Analysis of 148 kb of genomic DNA around the wnt1 locus of Fugu rubripes.

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Analysis of 148 kb of genomic DNA around the wnt1 locus of Fugu rubripes.

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