Fugu and human sequence comparison identifies novel human genes and conserved non-coding sequences

The compact genome of the pufferfish, Fugu rubripes, has been proposed as a 'reference' genome to aid in annotating and analysing the human genome. We have annotated and compared 85 kb of Fugu sequence containing 17 genes with its homologous loci in the human draft genome and identified three 'novel' human genes that were missed or incompletely predicted by the previous gene prediction methods. Two of the novel genes contain zinc finger domains and are designated ZNF366 and ZNF367. They map to human chromosomes 5q13.2 and 9q22.32, respectively. The third novel gene, designated C9orf21, maps to chromosome 9q22.32. This gene is unique to vertebrates, and the protein encoded by it does not contain any known domains. We could not find human homologs for two Fugu genes, a novel chemokine gene and a kinase gene. These genes are either specific to teleosts or lost in the human lineage. The Fugu-human comparison identified several conserved non-coding sequences in the promoter and intronic regions. These sequences, conserved during 450 million years of vertebrate evolution, are likely to be involved in gene regulation. The 85 kb Fugu locus is dispersed over four human loci, occupying about 1.5 Mb. Contiguity is conserved in the human genome between six out of 16 Fugu gene pairs. These contiguous chromosomal segments should share a common evolutionary history dating back to the common ancestor of mammals and teleosts. We propose contiguity as strong evidence to identify orthologous genes in distant organisms. This study confirms the utility of the Fugu as a supplementary tool to uncover and confirm novel genes and putative gene regulatory regions in the human genome.     

Biochemical implications of sequence comparisons of the cystic fibrosis transmembrane conductance regulator

System A, the Na(+)-dependent amino acid transport activity, is encoded by the ATA2 gene and up-regulated following partial hepatectomy (PH), and its competitive inhibition interferes with liver regeneration. Rabbit polyclonal antibody was raised against a portion of the ATA2 gene product followed by immunodetection of ATA2 in isolated liver plasma membrane and lysate. The level of ATA2 increased in the plasma membrane following PH, while the relatively high quantity of ATA2 found in liver lysate remained constant. We also have shown that Northern analysis of steady-state ATA2 mRNA revealed no significant change following PH. These data show that ATA2-mediated transport is not regulated by the steady-state level of ATA2 mRNA but is regulated by the amount of ATA2 and redistribution to the plasma membrane. We hypothesize that ATA2 activity is regulated by recruitment of ATA2 protein from an intracellular compartment. In addition, the pattern of expression of System A activity in oocytes, transport kinetics, and sensitivity to chemical modification indicate the presence of a second System A isoform in liver that differs substantially from ATA2.     

A genetic linkage map for the tiger pufferfish, Takifugu rubripes

The compact genome of the tiger pufferfish, Takifugu rubripes (fugu), has been sequenced to the "draft" level and annotated to identify all the genes. However, the assembly of the draft genome sequence is highly fragmented due to the lack of a genetic or a physical map. To determine the long-range linkage relationship of the sequences, we have constructed the first genetic linkage map for fugu. The maps for the male and female spanning 697.1 and 1213.5 cM, respectively, were arranged into 22 linkage groups by markers heterozygous in both parents. The resulting map consists of 200 microsatellite loci physically linked to genome sequences spanning approximately 39 Mb in total. Comparisons of the genome maps of fugu, other teleosts, and mammals suggest that syntenic relationship is more conserved in the teleost lineage than in the mammalian lineage. Map comparisons also show a pufferfish lineage-specific rearrangement of the genome resulting in colocalization of two Hox gene clusters in one linkage group. This map provides a foundation for development of a complete physical map, a basis for comparison of long-range linkage of genes with other vertebrates, and a resource for mapping loci responsible for phenotypic differences among Takifugu species.     

The Ancient Origins of Neural Substrates for Land Walking

Download video (10MB)Help with mp4 files     

Survey Sequencing and Comparative Analysis of the Elephant Shark (Callorhinchus milii) Genome

Owing to their phylogenetic position, cartilaginous fishes (sharks, rays, skates, and chimaeras) provide a critical reference for our understanding of vertebrate genome evolution. The relatively small genome of the elephant shark, Callorhinchus milii, a chimaera, makes it an attractive model cartilaginous fish genome for whole-genome sequencing and comparative analysis. Here, the authors describe survey sequencing (1.4× coverage) and comparative analysis of the elephant shark genome, one of the first cartilaginous fish genomes to be sequenced to this depth. Repetitive sequences, represented mainly by a novel family of short interspersed element–like and long interspersed element–like sequences, account for about 28% of the elephant shark genome. Fragments of approximately 15,000 elephant shark genes reveal specific examples of genes that have been lost differentially during the evolution of tetrapod and teleost fish lineages. Interestingly, the degree of conserved synteny and conserved sequences between the human and elephant shark genomes are higher than that between human and teleost fish genomes. Elephant shark contains putative four Hox clusters indicating that, unlike teleost fish genomes, the elephant shark genome has not experienced an additional whole-genome duplication. These findings underscore the importance of the elephant shark as a critical reference vertebrate genome for comparative analysis of the human and other vertebrate genomes. This study also demonstrates that a survey-sequencing approach can be applied productively for comparative analysis of distantly related vertebrate genomes.     

Neuropeptide Y-family peptides and receptors in the elephant shark, Callorhinchus milii confirm gene duplications before the gnathostome radiation

We describe here the repertoire of neuropeptide Y (NPY) peptides and receptors in the elephant shark Callorhinchus milii, belonging to the chondrichthyans that diverged from the rest of the gnathostome (jawed vertebrate) lineage about 450 million years ago and the first chondrichthyan with a genome project. We have identified two peptide genes that are orthologous to NPY and PYY (peptide YY) in other vertebrates, and seven receptor genes orthologous to the Y1, Y2, Y4, Y5, Y6, Y7 and Y8 subtypes found in tetrapods and teleost fishes. The repertoire of peptides and receptors seems to reflect the ancestral configuration in the predecessor of all gnathostomes, whereas other lineages such as mammals and teleosts have lost one or more receptor genes or have acquired 1-2 additional peptide genes. Both the peptides and receptors showed broad and overlapping mRNA expression which may explain why some receptor gene losses could take place in some lineages, but leaves open the question why all the known ancestral receptors have been retained in the elephant shark.     

Regulation of protocadherin gene expression by multiple neuron-restrictive silencer elements scattered in the gene cluster

The clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5′ intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.     

Functional conservation of a forebrain enhancer from the elephant shark (<Emphasis Type="Italic">Callorhinchus milii</Emphasis>) in zebrafish and mice

Background  

The phylogenetic position of the elephant shark (Callorhinchus milii ) is particularly relevant to study the evolution of genes and gene regulation in vertebrates. Here we examine the evolution of Dlx homeobox gene regulation during vertebrate embryonic development with a particular focus on the forebrain. We first identified the elephant shark sequence orthologous to the URE2 cis -regulatory element of the mouse Dlx1/Dlx2 locus (herein named CmURE2). We then conducted a comparative study of the sequence and enhancer activity of CmURE2 with that of orthologous regulatory sequences from zebrafish and mouse.