Humanity as the model system.
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Genetics, Medical↗
Biomedical subjects
Sydney Brenner
Publications and source records attributed to Sydney Brenner.
The regulation of retina specific expression of rhodopsin gene in vertebrates.
Rhodopsin genes in most vertebrate species, with the exception of teleost fishes, contain introns. Despite differences in the gene structure, similar regulatory motifs have been identified in fish, amphibian and mammalian rhodopsin promoters, suggesting that rhodopsin gene regulation may be conserved in vertebrates. However, there is no direct evidence to support this notion. To address this, the rhodopsin promoter from the pufferfish, Fugu rubripes, was isolated and tested in transgenic mice and frogs. A 6.5 kb Fugu genomic fragment containing the rhodopsin gene and 4.5 kb 5' flanking region was able to direct expression of the Fugu rhodopsin gene to the retina of transgenic mice. In transgenic tadpoles, photoreceptor rod cell-specific expression of a reporter gene was achieved using only 500 bp Fugu rhodopsin promoter fragment. Mutagenesis of this promoter fragment revealed that a conserved NRE-like motif is crucial for the retina-specific expression. Our investigation suggests that the regulation of retinal specific expression is conserved in the pufferfish, frog and mouse and that the ancestral intron-containing rhodopsin gene has been displaced by an intronless copy in teleosts.
Animals↗
Nature's gift to science (Nobel lecture).
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Animals↗
Hox gene clusters in the Indonesian coelacanth, Latimeria menadoensis.
The Hox genes encode transcription factors that play a key role in specifying body plans of metazoans. They are organized into clusters that contain up to 13 paralogue group members. The complex morphology of vertebrates has been attributed to the duplication of Hox clusters during vertebrate evolution. In contrast to the single Hox cluster in the amphioxus (Branchiostoma floridae), an invertebrate-chordate, mammals have four clusters containing 39 Hox genes. Ray-finned fishes (Actinopterygii) such as zebrafish and fugu possess more than four Hox clusters. The coelacanth occupies a basal phylogenetic position among lobe-finned fishes (Sarcopterygii), which gave rise to the tetrapod lineage. The lobe fins of sarcopterygians are considered to be the evolutionary precursors of tetrapod limbs. Thus, the characterization of Hox genes in the coelacanth should provide insights into the origin of tetrapod limbs. We have cloned the complete second exon of 33 Hox genes from the Indonesian coelacanth, Latimeria menadoensis, by extensive PCR survey and genome walking. Phylogenetic analysis shows that 32 of these genes have orthologs in the four mammalian HOX clusters, including three genes (HoxA6, D1, and D8) that are absent in ray-finned fishes. The remaining coelacanth gene is an ortholog of hoxc1 found in zebrafish but absent in mammals. Our results suggest that coelacanths have four Hox clusters bearing a gene complement more similar to mammals than to ray-finned fishes, but with an additional gene, HoxC1, which has been lost during the evolution of mammals from lobe-finned fishes.
Amino Acid Sequence↗
Molecular cloning of the pufferfish (Takifugu rubripes) Mx gene and functional characterization of its promoter.
Mx proteins are members of a family of interferon-inducible genes that are expressed by cells in response to viral infection. They are important determinants of innate immunity against viral infection in vertebrates. We cloned the pufferfish ( Takifugu rubripes) Mx gene and sequenced 80 kb from the Mx locus. The Fugu Mx gene spans 3.4 kb from the transcription start site to the polyadenylation signal, and is made up of 12 exons and 11 introns. The protein sequence encoded by the Fugu Mx gene is 77%, 48%, and 51% identical to that of trout Mx1, chicken Mx, and mouse Mx1 genes, respectively. The Fugu Mx gene is expressed in a variety of tissues, with high expression detected in the heart, gill, kidney, intestine, and brain. Analysis of the 5'-flanking sequence of the gene showed the presence of two interferon-stimulated response elements (ISRE) at positions -51 to 38 and -97 to 85, relative to the transcription start site. The Fugu Mx promoter was inducible by human IFN-beta in the human hepatoma (Huh7) cells and by polyinosinic: polycytidilic acid in the top minnow hepatoma (PLHC-1) cells. Deletion analysis of the promoter showed that both ISREs contributed to inducibility. These results demonstrate that the molecular mechanisms involved in Mx gene regulation are conserved between fish and mammals.
Amino Acid Sequence↗
Duplication, degeneration and subfunctionalization of the nested synapsin-Timp genes in Fugu.
The genes encoding synapsin (Syn) and the tissue inhibitor of metalloproteinase (Timp) exhibit a nested organization that is conserved in fruit fly and vertebrates. Analysis of the human and Fugu genomes show that the evolution of Syn-Timp gene families is characterized by duplications, secondary loss and the partitioning of ancestral functions. Of particular interest are two duplicate Syn-Timp loci in Fugu that have accumulated complementary degenerate mutations such that each Syn duplicate produces one of the two transcripts generated from the single ancestral gene, and one of the Timp genes is lost.
Alternative Splicing↗
Worms and science. An interview with Sydney Brenner, distinguished research professor at The Salk Institute, La Jolla, USA, and one of the winners of 2002 Nobel Prize for Physiology and Medicine.
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Animals↗
Inducible system in human hepatoma cell lines for hepatitis C virus production.
We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetracycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 h to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect nai;ve Huh7 and stable Huh7-human CD81 cells.
Carcinoma, Hepatocellular↗
Identification and analysis of additional copies of the platelet-derived growth factor receptor and colony stimulating factor 1 receptor genes in fugu.
The receptors for the platelet-derived growth factor (PDGFRalpha and PDGFRbeta) belong to a subfamily of protein tyrosine kinase receptors that also includes kit and the colony stimulating factor-1 receptor (CSF1R). In mammals, the genes encoding PDGFRalpha and PDGFRbeta are tandemly linked to the kit and CSF1R genes, respectively. Based on the structural similarity and genomic organization of these four genes, it has been suggested that they arose from an ancestral protein tyrosine kinase receptor gene by two rounds of duplication. We have previously cloned the PDGFRbeta and CSF1R genes from the pufferfish, Fugu rubripes, and shown that they are tandemly linked like the mammalian genes [Genome Res. 6 (1996) 1185]. We have now cloned two additional members of this gene family, fPDGFRbeta2 and fCSF1R2 from the fugu and shown that these two genes are also tandemly linked. This indicates that the PDGFRbeta-CSF1R locus has been duplicated in the lineage leading to fugu. The fugu fPDGFRbeta2 and fCSF1R2 genes contain three and one extra introns, respectively, compared with other members of this family. Polymerase chain reaction cloning of a conserved region of PDGFRbeta gene from other ray-finned fishes identified two copies in the zebrafish (order Cypriniformes) and sunfish (order Tetraodontiformes). These results are discussed in the context of the proposed teleost lineage-specific whole genome duplication hypothesis.
Amino Acid Sequence↗
Whole-genome shotgun assembly and analysis of the genome of Fugu rubripes.
The compact genome of Fugu rubripes has been sequenced to over 95% coverage, and more than 80% of the assembly is in multigene-sized scaffolds. In this 365-megabase vertebrate genome, repetitive DNA accounts for less than one-sixth of the sequence, and gene loci occupy about one-third of the genome. As with the human genome, gene loci are not evenly distributed, but are clustered into sparse and dense regions. Some "giant" genes were observed that had average coding sequence sizes but were spread over genomic lengths significantly larger than those of their human orthologs. Although three-quarters of predicted human proteins have a strong match to Fugu, approximately a quarter of the human proteins had highly diverged from or had no pufferfish homologs, highlighting the extent of protein evolution in the 450 million years since teleosts and mammals diverged. Conserved linkages between Fugu and human genes indicate the preservation of chromosomal segments from the common vertebrate ancestor, but with considerable scrambling of gene order.
Animals↗
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.
Amino Acid Sequence↗
Life sentences: elementary zenetics--do or dai.
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Buddhism↗
Life sentences: Ontology recapitulates philology.
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Molecular Biology↗
Conserved regulation of the lymphocyte-specific expression of lck in the Fugu and mammals.
The lck gene encodes a lymphocyte-specific protein-tyrosine kinase that is implicated in T cell maturation and signaling. In mammals, the transcription of the lck gene is regulated by two independent promoters, the proximal promoter, which is active in thymocytes, and the distal promoter, which dominates in mature T cells. In the human and mouse lck gene loci, the two promoter elements are separated by at least 40 kb and 10 kb, respectively. In this study, we have cloned and sequenced 60 kb from the pufferfish (Fugu rubripes) lck locus. The promoter region of the Fugu lck spans only 4.2 kb and contains a proximal and a distal promoter in the 2.3-kb region adjacent to the coding sequence. By generating transgenic mice, we have demonstrated that the compact promoter of the Fugu lck contains regulatory elements that direct expression to lymphoid organs of mice. We were able to localize the regulatory elements to a short region of 830 bp without losing specificity to cultured human T cell line. These results show that the basic mechanisms that mediate lymphocyte-specific expression are conserved between teleosts and mammals. The short promoter of the Fugu lck isolated by us offers a powerful tool for labeling T cells, targeting expression, and manipulating T cell activity in fishes as well as in mammals.
Amino Acid Sequence↗
Novel vertebrate genes and putative regulatory elements identified at kidney disease and NR2E1/fierce loci.
Fierce (frc) mice are deleted for nuclear receptor 2e1 (Nr2e1), and exhibit cerebral hypoplasia, blindness, and extreme aggression. To characterize the Nr2e1 locus, which may also contain the mouse kidney disease (kd) allele, we compared sequence from human, mouse, and the puffer fish Fugu rubripes. We identified a novel gene, c222389, containing conserved elements in noncoding regions. We also discovered a novel vertebrate gene conserved across its length in prokaryotes and invertebrates. Based on a dramatic upregulation in lactating breast, we named this gene lactation elevated-1 (LACE1). Two separate 100-bp elements within the first NR2E1 intron were virtually identical between the three species, despite an estimated 450 million years of divergent evolution. These elements represent strong candidates for functional NR2E1 regulatory elements in vertebrates. A high degree of conservation across NR2E1 combined with a lack of interspersed repeats suggests that an array of regulatory elements embedded within the gene is required for proper gene expression.
Alternative Splicing↗
Characterization of the platelet-derived growth factor receptor alpha and c-kit genes in the pufferfish Fugu rubripes.
The receptor tyrosine kinase type III subfamily includes the PDGF receptors (alpha and beta), c-kit and the colony stimulating factor 1 receptor (CSF1R). The similar genomic organization of the genes encoding these receptors and the observation that in mammals, the genes for PDGFR alpha and PDGFR beta are tandemly linked with the c-kit and CSF1R genes, respectively, suggest that these genes arose from an ancestral receptor tyrosine kinase gene. We have previously cloned two copies each of the PDGFR beta and CSF1R genes from the pufferfish, Fugu rubripes, and shown that they are tandemly linked. We have now cloned the Fugu PDGFR alpha and c-kit genes and show that they are also tandemly linked. The intergenic region between the two genes in the Fugu (13 kb) and humans (350 kb) contains a conserved 59 bp element, which may play a role in regulation. The Fugu PDGFR alpha gene is transcribed from two promoters giving rise to alternate transcripts.
Amino Acid Sequence↗
Massively parallel signature sequencing (MPSS) as a tool for in-depth quantitative gene expression profiling in all organisms.
Massively parallel signature sequencing (MPSS) is one of the newest tools available for conducting in-depth expression profiling. MPSS is an open-ended platform that analyses the level of expression of virtually all genes in a sample by counting the number of individual mRNA molecules produced from each gene. There is no requirement that genes be identified and characterised prior to conducting an experiment. MPSS has a routine sensitivity at a level of a few molecules of mRNA per cell, and the datasets are in a digital format that simplifies the management and analysis of the data. Therefore, of the various microarray and non-microarray technologies currently available, MPSS provides many advantages for generating the type of complete datasets that will help to facilitate hypothesis-driven experiments in the era of digital biology.
Cloning, Molecular↗