Generation and Analysis of 25 Mb of Genomic DNA from the Pufferfish Fugu rubripes by Sequence Scanning

Abstract

We have generated and analyzed >50,000 shotgun clones from 1059 Fugu cosmid clones. All sequences have been minimally edited and searched against protein and DNA databases. These data are all displayed on a searchable, publicly available web site at http://fugu.hgmp.mrc.ac.uk/. With an average of 50 reads per cosmid, this is virtually nonredundant sequence skimming, covering 30%-50% of each clone. This essentially random data set covers nearly 25 Mb (>6%) of the Fugu genome and forms the basis of a series of whole genome analyses which address questions regarding gene density and distribution in the Fugu genome and the similarity between Fugu and mammalian genes. The Fugu genome, with eight times less DNA but a similar gene repertoire, is ideally suited to this type of study because most cosmids contain more than one identifiable gene. General features of the genome are also discussed. We have made some estimation of the syntenic relationship between mammals and Fugu and looked at the efficacy of ORF prediction from short, unedited Fugu genomic sequences. Comparative DNA sequence analyses are an essential tool in the functional interpretation of complex vertebrate genomes. This project highlights the utility of using the Fugu genome in this kind of study.Despite massive investment in genome mapping and DNA sequencing over the last 10 years, large-scale sequencing of vertebrate genomes has been initiated only very recently. This is partly because the initial emphasis has been on developing mapping, sequencing, and assembly technologies and partly because sequence-ready contigs of large regions of the human genome have not been available. Many valuable lessons have been learned-at no small expense-from the bacterial, yeast, and, in particular, the Caenorhabditis elegans projects. It is also clear, however, that mammalian genomes may present additional problems relating to the generation of cloned DNA from some regions, sequence assembly of highly repetitive DNA, and the large size of the genomes involved.To interpret much of the data, comparative sequencing of genomic regions from other vertebrates will be necessary. The identification of conserved sequences across species has always been a key technique in the identification of genes. In addition, sequence comparison in invertebrate projects has identified many genes by sequence similarity and in many cases has allowed speculation on function. Now that the resolution of genomes is approaching the single base pair, powerful analytical methods need to be used to define the many elements-both coding and noncoding-that are contained within the human genome.Despite the need for comparison, there is little investment in other vertebrate sequencing projects at this time. Small regions of conserved synteny within the mouse genome have been pinpointed for complete genomic sequencing, and this will provide an opportunity to compare not only precise orders of genes but also regions in and around the coding sequence itself. This should lead to the identification of other con...

Regulation of the stem cell leukemia ( SCL ) gene: A tale of two fishes

“…7, which is published as supplemental data on the PNAS web site, www.pnas.org). These data are consistent with previous observations that, in at least some regions of the genome, pufferfish speciation has been accompanied by particularly high rates of chromosomal rearrangements (61)(62)(63)(64). It is possible that genome compression in the pufferfish has been achieved, at least in part, by clustering together genes with shared domains of expression.…”

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

“…During routine Fugu cosmid library sequence scanning (within the confines of the Fugu Landmark Mapping Project (Elgar et al 1999)) a cosmid (C139D21) was identified as having a gene fragment with high sequence similarity to goldfish VPAC 1 R (probability value in excess of 9e -19 ). PCR primers were designed for a conserved sequence motif of this family identified after a multiple sequence alignment of all the members of this family and the Fugu cosmid C139D21 partial sequence.…”

The Ghost of Selection Past: Rates of Evolution and Functional Divergence of Anciently Duplicated Genes

“…For example, Morizot et al (1991) have estimated that the genome of the platyfish (genus Xiphophorus) is five times smaller than the human genome. Elgar et al (1999) estimated that the pufferfish genome is eight times smaller than the human genome and suggested that the duplication of Hox clusters by regional duplication is easier to reconcile with fish genome size data than a complete genome duplication. However, the small genome of Fugu may be due to the reduction of intergenic and noncoding regions (Venkatesh et al 2000).…”