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1.
Effective targeted gene 'knockdown' in zebrafish 总被引:26,自引:0,他引:26
The sequencing of the zebrafish genome should be completed by the end of 2002. Direct assignment of function on the basis of this information would be facilitated by the development of a rapid, targeted 'knockdown' technology in this model vertebrate. We show here that antisense, morpholino-modified oligonucleotides (morpholinos) are effective and specific translational inhibitors in zebrafish. We generated phenocopies of mutations of the genes no tail (ref. 2), chordin (ref. 3), one-eyed-pinhead (ref. 4), nacre (ref. 5) and sparse (ref. 6), removing gene function from maternal through post-segmentation and organogenesis developmental stages. We blocked expression from a ubiquitous green fluorescent protein (GFP) transgene, showing that, unlike tissue-restricted limitations found with RNA-based interference in the nematode, all zebrafish cells readily respond to this technique. We also developed also morpholino-based zebrafish models of human disease. Morpholinos targeted to the uroporphyrinogen decarboxylase gene result in embryos with hepatoerythropoietic porphyria. We also used morpholinos for the determination of new gene functions. We showed that embryos with reduced sonic hedgehog (ref. 9) signalling and reduced tiggy-winkle hedgehog (ref. 10) function exhibit partial cyclopia and other specific midline abnormalities, providing a zebrafish genetic model for the common human disorder holoprosencephaly. Conserved vertebrate processes and diseases are now amenable to a systematic, in vivo, reverse-genetic paradigm using zebrafish embryos. 相似文献
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We describe a new synthetic lethality analysis by microarray (SLAM) technique that uses approximately 4,600 Saccharomyces cerevisiae haploid deletion mutants with molecular 'bar codes' (TAGs). We used SGS1 and SRS2, two 3'-->5' DNA helicase genes, as 'queries' to identify their redundant and unique biological functions. We introduced these 'query mutations' into a haploid deletion pool by integrative transformation to disrupt the query gene in every cell, generating a double mutant pool. Optimization of integrative transformation efficiency was essential to the success of SLAM. Synthetic interactions defined a DNA helicase genetic network and predicted a role for SRS2 in processing damaged replication forks but, unlike SGS1, not in rDNA replication, DNA topology or lagging strand synthesis. SGS1 and SRS2 have synthetic defects with MRC1 but not RAD9, suggesting that SGS1 and SRS2 function in a parallel pathway with MRC1 to transduce the DNA replication stress signal to the general DNA damage checkpoint pathway. Both helicase genes have rad51-reversible synthetic defects with 5'-->3' DNA helicase RRM3, suggesting that RRM3 helps prevent formation of toxic recombination intermediates. SLAM detects synthetic lethality efficiently and ranks candidate genetic interactions, making it an especially useful method. 相似文献
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Roest Crollius H Jaillon O Bernot A Dasilva C Bouneau L Fischer C Fizames C Wincker P Brottier P Quétier F Saurin W Weissenbach J 《Nature genetics》2000,25(2):235-238
The number of genes in the human genome is unknown, with estimates ranging from 50,000 to 90,000 (refs 1, 2), and to more than 140,000 according to unpublished sources. We have developed 'Exofish', a procedure based on homology searches, to identify human genes quickly and reliably. This method relies on the sequence of another vertebrate, the pufferfish Tetraodon nigroviridis, to detect conserved sequences with a very low background. Similar to Fugu rubripes, a marine pufferfish proposed by Brenner et al. as a model for genomic studies, T. nigroviridis is a more practical alternative with a genome also eight times more compact than that of human. Many comparisons have been made between F. rubripes and human DNA that demonstrate the potential of comparative genomics using the pufferfish genome. Application of Exofish to the December version of the working draft sequence of the human genome and to Unigene showed that the human genome contains 28,000-34,000 genes, and that Unigene contains less than 40% of the protein-coding fraction of the human genome. 相似文献
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Chromatin packaging in mammalian spermatozoa requires an ordered replacement of the somatic histones by two classes of spermatid-specific basic proteins, the transition proteins and the protamines. Temporal expression of transition proteins and protamines during spermatid differentiation is under translational control, and premature translation of protamine 1 leads to precocious nuclear condensation and sterility. We have previously suggested that the double-stranded (ds) RNA binding protein Prbp (encoded by the gene Tarbp2) functions as a translational regulator during mouse spermatogenesis. Here we show that Prbp is required for proper translational activation of the mRNAs encoding the protamines. We generated mice that carry a targeted disruption of Tarbp2 and determined that they were sterile and severely oligospermic. Using immunohistological analysis, we determined that the endogenous Prm2 mRNA and a reporter mRNA carrying protamine 1 translational-control elements were translated in a mosaic pattern. We showed that failure to synthesize the protamines resulted in delayed replacement of the transition proteins and subsequent failure of spermiation. The timing of Prbp expression suggests that it may function as a chaperone in the assembly of specific translationally regulated ribonucleoprotein particles. 相似文献
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Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation 总被引:1,自引:0,他引:1
The hindbrain roof plate and choroid plexus are essential organizing centers for inducing dorsal neuron fates and sustaining neuron function. To map the formation of these structures, we developed a broadly applicable, high resolution, recombinase-based method for mapping the fate of cells originating from coordinates defined by intersecting combinations of expressed genes. Using this method, we show that distinct regions of hindbrain roof plate originate from discrete subdomains of rhombencephalic neuroectoderm expressing Wnt1; that choroid plexus, a secretory epithelium important for patterning later-formed hindbrain structures and maintaining neuron function, derives from the same embryonic primordium as the hindbrain roof plate; and that, unlike the floor plate, these dorsal organizing centers develop in a patterned, segmental manner, built from lineage-restricted compartments. Our data suggest that the roof plate and choroid plexus may be formed of functional units that are capable of differentially organizing the generation of distinct neuronal cell types at different axial levels. 相似文献
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Wan J Yourshaw M Mamsa H Rudnik-Schöneborn S Menezes MP Hong JE Leong DW Senderek J Salman MS Chitayat D Seeman P von Moers A Graul-Neumann L Kornberg AJ Castro-Gago M Sobrido MJ Sanefuji M Shieh PB Salamon N Kim RC Vinters HV Chen Z Zerres K Ryan MM Nelson SF Jen JC 《Nature genetics》2012,44(6):704-708
RNA exosomes are multi-subunit complexes conserved throughout evolution and are emerging as the major cellular machinery for processing, surveillance and turnover of a diverse spectrum of coding and noncoding RNA substrates essential for viability. By exome sequencing, we discovered recessive mutations in EXOSC3 (encoding exosome component 3) in four siblings with infantile spinal motor neuron disease, cerebellar atrophy, progressive microcephaly and profound global developmental delay, consistent with pontocerebellar hypoplasia type 1 (PCH1; MIM 607596). We identified mutations in EXOSC3 in an additional 8 of 12 families with PCH1. Morpholino knockdown of exosc3 in zebrafish embryos caused embryonic maldevelopment, resulting in small brain size and poor motility, reminiscent of human clinical features, and these defects were largely rescued by co-injection with wild-type but not mutant exosc3 mRNA. These findings represent the first example of an RNA exosome core component gene that is responsible for a human disease and further implicate dysregulation of RNA processing in cerebellar and spinal motor neuron maldevelopment and degeneration. 相似文献
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The deoxyguanosine kinase gene is mutated in individuals with depleted hepatocerebral mitochondrial DNA. 总被引:19,自引:0,他引:19
H Mandel R Szargel V Labay O Elpeleg A Saada A Shalata Y Anbinder D Berkowitz C Hartman M Barak S Eriksson N Cohen 《Nature genetics》2001,29(3):337-341
Mitochondrial DNA (mtDNA)-depletion syndromes (MDS; OMIM 251880) are phenotypically heterogeneous, autosomal-recessive disorders characterized by tissue-specific reduction in mtDNA copy number. Affected individuals with the hepatocerebral form of MDS have early progressive liver failure and neurological abnormalities, hypoglycemia and increased lactate in body fluids. Affected tissues show both decreased activity of the mtDNA-encoded respiratory chain complexes (I, III, IV, V) and mtDNA depletion. We used homozygosity mapping in three kindreds of Druze origin to map the gene causing hepatocerebral MDS to a region of 6.1 cM on chromosome 2p13, between markers D2S291 and D2S2116. This interval encompasses the gene (DGUOK) encoding the mitochondrial deoxyguanosine kinase (dGK). We identified a single-nucleotide deletion (204delA) within the coding region of DGUOK that segregates with the disease in the three kindreds studied. Western-blot analysis did not detect dGK protein in the liver of affected individuals. The main supply of deoxyribonucleotides (dNTPs) for mtDNA synthesis comes from the salvage pathway initiated by dGK and thymidine kinase-2 (TK2). The association of mtDNA depletion with mutated DGUOK suggests that the salvage-pathway enzymes are involved in the maintenance of balanced mitochondrial dNTP pools. 相似文献
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Single nucleotide polymorphisms (SNPs) are valuable genetic markers of human disease. They also comprise the highest potential density marker set available for mapping experimentally derived mutations in model organisms such as Caenorhabditis elegans. To facilitate the positional cloning of mutations we have identified polymorphisms in CB4856, an isolate from a Hawaiian island that shows a uniformly high density of polymorphisms compared with the reference Bristol N2 strain. Based on 5.4 Mbp of aligned sequences, we predicted 6,222 polymorphisms. Furthermore, 3,457 of these markers modify restriction enzyme recognition sites ('snip-SNPs') and are therefore easily detected as RFLPs. Of these, 493 were experimentally confirmed by restriction digest to produce a snip-SNP map of the worm genome. A mapping strategy using snip-SNPs and bulked segregant analysis (BSA) is outlined. CB4856 is crossed into a mutant strain, and exclusion of CB4856 alleles of a subset of snip-SNPs in mutant progeny is assessed with BSA. The proximity of a linked marker to the mutation is estimated by the relative proportion of each form of the biallelic marker in populations of wildtype and mutant genomes. The usefulness of this approach is illustrated by the rapid mapping of the dyf-5 gene. 相似文献
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Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. 总被引:22,自引:0,他引:22
J N Spelbrink F Y Li V Tiranti K Nikali Q P Yuan M Tariq S Wanrooij N Garrido G Comi L Morandi L Santoro A Toscano G M Fabrizi H Somer R Croxen D Beeson J Poulton A Suomalainen H T Jacobs M Zeviani C Larsson 《Nature genetics》2001,28(3):223-231
The gene products involved in mammalian mitochondrial DNA (mtDNA) maintenance and organization remain largely unknown. We report here a novel mitochondrial protein, Twinkle, with structural similarity to phage T7 gene 4 primase/helicase and other hexameric ring helicases. Twinkle colocalizes with mtDNA in mitochondrial nucleoids. Screening of the gene encoding Twinkle in individuals with autosomal dominant progressive external ophthalmoplegia (adPEO), associated with multiple mtDNA deletions, identified 11 different coding-region mutations co-segregating with the disorder in 12 adPEO pedigrees of various ethnic origins. The mutations cluster in a region of the protein proposed to be involved in subunit interactions. The function of Twinkle is inferred to be critical for lifetime maintenance of human mtDNA integrity. 相似文献
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Exome sequencing identifies somatic mutations of DNA methyltransferase gene DNMT3A in acute monocytic leukemia 总被引:1,自引:0,他引:1
Yan XJ Xu J Gu ZH Pan CM Lu G Shen Y Shi JY Zhu YM Tang L Zhang XW Liang WX Mi JQ Song HD Li KQ Chen Z Chen SJ 《Nature genetics》2011,43(4):309-315
Abnormal epigenetic regulation has been implicated in oncogenesis. We report here the identification of somatic mutations by exome sequencing in acute monocytic leukemia, the M5 subtype of acute myeloid leukemia (AML-M5). We discovered mutations in DNMT3A (encoding DNA methyltransferase 3A) in 23 of 112 (20.5%) cases. The DNMT3A mutants showed reduced enzymatic activity or aberrant affinity to histone H3 in vitro. Notably, there were alterations of DNA methylation patterns and/or gene expression profiles (such as HOXB genes) in samples with DNMT3A mutations as compared with those without such changes. Leukemias with DNMT3A mutations constituted a group of poor prognosis with elderly disease onset and of promonocytic as well as monocytic predominance among AML-M5 individuals. Screening other leukemia subtypes showed Arg882 alterations in 13.6% of acute myelomonocytic leukemia (AML-M4) cases. Our work suggests a contribution of aberrant DNA methyltransferase activity to the pathogenesis of acute monocytic leukemia and provides a useful new biomarker for relevant cases. 相似文献
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Somatic integration and long-term transgene expression in normal and haemophilic mice using a DNA transposon system 总被引:30,自引:0,他引:30
The development of non-viral gene-transfer technologies that can support stable chromosomal integration and persistent gene expression in vivo is desirable. Here we describe the successful use of transposon technology for the nonhomologous insertion of foreign genes into the genomes of adult mammals using naked DNA. We show that the Sleeping Beauty transposase can efficiently insert transposon DNA into the mouse genome in approximately 5-6% of transfected mouse liver cells. Chromosomal transposition resulted in long-term expression (>5 months) of human blood coagulation factor IX at levels that were therapeutic in a mouse model of haemophilia B. Our results establish DNA-mediated transposition as a new genetic tool for mammals, and provide new strategies to improve existing non-viral and viral vectors for human gene therapy applications. 相似文献
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Breakage-fusion-bridge cycles contribute to chromosome instability and generate large DNA palindromes that facilitate gene amplification in human cancers. The prevalence of large DNA palindromes in cancer is not known. Here, by using a new microarray-based approach called genome-wide analysis of palindrome formation, we show that palindromes occur frequently and are widespread in human cancers. Individual tumors seem to have a nonrandom distribution of palindromes in their genomes, and a subset of palindromic loci is associated with gene amplification. This indicates that the location of palindromes in the cancer genome can serve as a structural platform that supports subsequent gene amplification. Genome-wide analysis of palindrome formation is a new approach to identify structural chromosome aberrations associated with cancer. 相似文献
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To identify expressed sequences within candidate regions for the Huntington's disease (HD) gene in 4p16.3, we isolated the gene encoding the beta subunit of the human cGMP phosphodiesterase (PDEB). We formally assessed this as a candidate gene for HD based on it's expression in brain, the demonstration of linkage disequilibrium between intragenic DNA markers and HD, and the demonstration that mice with a mutation in this gene have a reduction of neurons in particular brain regions. We investigated all 22 exons of PDEB and 5'-flanking region for point mutations in 16 HD patients of different ethnic origins using single strand conformational polymorphism analysis. The underlying DNA changes found initially exclusively in HD patients were excluded as the cause for HD. 相似文献
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The DNA repair gene MBD4 (MED1) is mutated in human carcinomas with microsatellite instability. 总被引:12,自引:0,他引:12
A Riccio L A Aaltonen A K Godwin A Loukola A Percesepe R Salovaara V Masciullo M Genuardi M Paravatou-Petsotas D E Bassi B A Ruggeri A J Klein-Szanto J R Testa G Neri A Bellacosa 《Nature genetics》1999,23(3):266-268
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Mutations in the gene encoding the 3'-5' DNA exonuclease TREX1 are associated with systemic lupus erythematosus 总被引:1,自引:0,他引:1
Lee-Kirsch MA Gong M Chowdhury D Senenko L Engel K Lee YA de Silva U Bailey SL Witte T Vyse TJ Kere J Pfeiffer C Harvey S Wong A Koskenmies S Hummel O Rohde K Schmidt RE Dominiczak AF Gahr M Hollis T Perrino FW Lieberman J Hübner N 《Nature genetics》2007,39(9):1065-1067
TREX1 acts in concert with the SET complex in granzyme A-mediated apoptosis, and mutations in TREX1 cause Aicardi-Goutières syndrome and familial chilblain lupus. Here, we report monoallelic frameshift or missense mutations and one 3' UTR variant of TREX1 present in 9/417 individuals with systemic lupus erythematosus but absent in 1,712 controls (P = 4.1 x 10(-7)). We demonstrate that two mutant TREX1 alleles alter subcellular targeting. Our findings implicate TREX1 in the pathogenesis of SLE. 相似文献