Publications by authors named "Ian Sealy"

15 Publications

  • Page 1 of 1

Common and distinct transcriptional signatures of mammalian embryonic lethality.

Nat Commun 2019 06 26;10(1):2792. Epub 2019 Jun 26.

Wellcome Sanger Institute, Wellcome Genome Campus, Cambridge, CB10 1SA, UK.

The Deciphering the Mechanisms of Developmental Disorders programme has analysed the morphological and molecular phenotypes of embryonic and perinatal lethal mouse mutant lines in order to investigate the causes of embryonic lethality. Here we show that individual whole-embryo RNA-seq of 73 mouse mutant lines (>1000 transcriptomes) identifies transcriptional events underlying embryonic lethality and associates previously uncharacterised genes with specific pathways and tissues. For example, our data suggest that Hmgxb3 is involved in DNA-damage repair and cell-cycle regulation. Further, we separate embryonic delay signatures from mutant line-specific transcriptional changes by developing a baseline mRNA expression catalogue of wild-type mice during early embryogenesis (4-36 somites). Analysis of transcription outside coding sequence identifies deregulation of repetitive elements in Morc2a mutants and a gene involved in gene-specific splicing. Collectively, this work provides a large scale resource to further our understanding of early embryonic developmental disorders.
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http://dx.doi.org/10.1038/s41467-019-10642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594971PMC
June 2019

The gene regulatory basis of genetic compensation during neural crest induction.

PLoS Genet 2019 06 14;15(6):e1008213. Epub 2019 Jun 14.

Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

The neural crest (NC) is a vertebrate-specific cell type that contributes to a wide range of different tissues across all three germ layers. The gene regulatory network (GRN) responsible for the formation of neural crest is conserved across vertebrates. Central to the induction of the NC GRN are AP-2 and SoxE transcription factors. NC induction robustness is ensured through the ability of some of these transcription factors to compensate loss of function of gene family members. However the gene regulatory events underlying compensation are poorly understood. We have used gene knockout and RNA sequencing strategies to dissect NC induction and compensation in zebrafish. We genetically ablate the NC using double mutants of tfap2a;tfap2c or remove specific subsets of the NC with sox10 and mitfa knockouts and characterise genome-wide gene expression levels across multiple time points. We find that compensation through a single wild-type allele of tfap2c is capable of maintaining early NC induction and differentiation in the absence of tfap2a function, but many target genes have abnormal expression levels and therefore show sensitivity to the reduced tfap2 dosage. This separation of morphological and molecular phenotypes identifies a core set of genes required for early NC development. We also identify the 15 somites stage as the peak of the molecular phenotype which strongly diminishes at 24 hpf even as the morphological phenotype becomes more apparent. Using gene knockouts, we associate previously uncharacterised genes with pigment cell development and establish a role for maternal Hippo signalling in melanocyte differentiation. This work extends and refines the NC GRN while also uncovering the transcriptional basis of genetic compensation via paralogues.
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http://dx.doi.org/10.1371/journal.pgen.1008213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6594659PMC
June 2019

Compensatory growth renders Tcf7l1a dispensable for eye formation despite its requirement in eye field specification.

Elife 2019 02 19;8. Epub 2019 Feb 19.

Department of Cell and Developmental Biology, University College London, London, United Kingdom.

The vertebrate eye originates from the eye field, a domain of cells specified by a small number of transcription factors. In this study, we show that Tcf7l1a is one such transcription factor that acts cell-autonomously to specify the eye field in zebrafish. Despite the much-reduced eye field in mutants, these fish develop normal eyes revealing a striking ability of the eye to recover from a severe early phenotype. This robustness is not mediated through genetic compensation at neural plate stage; instead, the smaller optic vesicle of mutants shows delayed neurogenesis and continues to grow until it achieves approximately normal size. Although the developing eye is robust to the lack of Tcf7l1a function, it is sensitised to the effects of additional mutations. In support of this, a forward genetic screen identified mutations in , and , which give synthetically enhanced eye specification or growth phenotypes when in combination with the mutation.
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http://dx.doi.org/10.7554/eLife.40093DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6380838PMC
February 2019

The age of heterozygous mutant parents influences the adult phenotype of their offspring irrespective of genotype in zebrafish.

Wellcome Open Res 2017 4;2:77. Epub 2017 Sep 4.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, UK.

Background: Mutations in proteins involved in telomere maintenance lead to a range of human diseases, including dyskeratosis congenita, idiopathic pulmonary fibrosis and cancer. Telomerase functions to add telomeric repeats back onto the ends of chromosomes, however non-canonical roles of components of telomerase have recently been suggested.

Methods: Here we use a zebrafish telomerase mutant which harbours a nonsense mutation in to investigate the adult phenotypes of fish derived from heterozygous parents of different ages. Furthermore we use whole genome sequencing data to estimate average telomere lengths.

Results: We show that homozygous offspring from older heterozygotes exhibit signs of body wasting at a younger age than those of younger parents, and that offspring of older heterozygous parents weigh less irrespective of genotype. We also demonstrate that homozygous mutant fish have a male sex bias, and that clutches from older parents also have a male sex bias in the heterozygous and wild-type populations. Telomere length analysis reveals that the telomeres of younger heterozygous parents are shorter than those of older heterozygous parents.

Conclusions: These data indicate that the phenotypes observed in offspring from older parents cannot be explained by telomere length. Instead we propose that Tert functions outside of telomere length maintenance in an age-dependent manner to influence the adult phenotypes of the next generation.
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http://dx.doi.org/10.12688/wellcomeopenres.12530.2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5840683PMC
September 2017

A high-resolution mRNA expression time course of embryonic development in zebrafish.

Elife 2017 11 16;6. Epub 2017 Nov 16.

Wellcome Trust Sanger Institute, Hinxton, United Kingdom.

We have produced an mRNA expression time course of zebrafish development across 18 time points from 1 cell to 5 days post-fertilisation sampling individual and pools of embryos. Using poly(A) pulldown stranded RNA-seq and a 3' end transcript counting method we characterise temporal expression profiles of 23,642 genes. We identify temporal and functional transcript co-variance that associates 5024 unnamed genes with distinct developmental time points. Specifically, a class of over 100 previously uncharacterised zinc finger domain containing genes, located on the long arm of chromosome 4, is expressed in a sharp peak during zygotic genome activation. In addition, the data reveal new genes and transcripts, differential use of exons and previously unidentified 3' ends across development, new primary microRNAs and temporal divergence of gene paralogues generated in the teleost genome duplication. To make this dataset a useful baseline reference, the data can be browsed and downloaded at Expression Atlas and Ensembl.
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http://dx.doi.org/10.7554/eLife.30860DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5690287PMC
November 2017

Loss of the chromatin modifier Kdm2aa causes BrafV600E-independent spontaneous melanoma in zebrafish.

PLoS Genet 2017 Aug 14;13(8):e1006959. Epub 2017 Aug 14.

Wellcome Trust Sanger Institute, Wellcome Genome Campus, Hinxton, United Kingdom.

KDM2A is a histone demethylase associated with transcriptional silencing, however very little is known about its in vivo role in development and disease. Here we demonstrate that loss of the orthologue kdm2aa in zebrafish causes widespread transcriptional disruption and leads to spontaneous melanomas at a high frequency. Fish homozygous for two independent premature stop codon alleles show reduced growth and survival, a strong male sex bias, and homozygous females exhibit a progressive oogenesis defect. kdm2aa mutant fish also develop melanomas from early adulthood onwards which are independent from mutations in braf and other common oncogenes and tumour suppressors as revealed by deep whole exome sequencing. In addition to effects on translation and DNA replication gene expression, high-replicate RNA-seq in morphologically normal individuals demonstrates a stable regulatory response of epigenetic modifiers and the specific de-repression of a group of zinc finger genes residing in constitutive heterochromatin. Together our data reveal a complex role for Kdm2aa in regulating normal mRNA levels and carcinogenesis. These findings establish kdm2aa mutants as the first single gene knockout model of melanoma biology.
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http://dx.doi.org/10.1371/journal.pgen.1006959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5570503PMC
August 2017

gEVAL - a web-based browser for evaluating genome assemblies.

Bioinformatics 2016 08 7;32(16):2508-10. Epub 2016 Apr 7.

Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Motivation: For most research approaches, genome analyses are dependent on the existence of a high quality genome reference assembly. However, the local accuracy of an assembly remains difficult to assess and improve. The gEVAL browser allows the user to interrogate an assembly in any region of the genome by comparing it to different datasets and evaluating the concordance. These analyses include: a wide variety of sequence alignments, comparative analyses of multiple genome assemblies, and consistency with optical and other physical maps. gEVAL highlights allelic variations, regions of low complexity, abnormal coverage, and potential sequence and assembly errors, and offers strategies for improvement. Although gEVAL focuses primarily on sequence integrity, it can also display arbitrary annotation including from Ensembl or TrackHub sources. We provide gEVAL web sites for many human, mouse, zebrafish and chicken assemblies to support the Genome Reference Consortium, and gEVAL is also downloadable to enable its use for any organism and assembly.

Availability And Implementation: Web Browser: http://geval.sanger.ac.uk, Plugin: http://wchow.github.io/wtsi-geval-plugin

Contact: [email protected]

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btw159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4978925PMC
August 2016

Genome-wide mapping of Hif-1α binding sites in zebrafish.

BMC Genomics 2015 Nov 11;16:923. Epub 2015 Nov 11.

Bateson Centre, Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, UK.

Background: Hypoxia Inducible Factor (HIF) regulates a cascade of transcriptional events in response to decreased oxygenation, acting from the cellular to the physiological level. This response is evolutionarily conserved, allowing the use of zebrafish (Danio rerio) as a model for studying the hypoxic response. Activation of the hypoxic response can be achieved in zebrafish by homozygous null mutation of the von Hippel-Lindau (vhl) tumour suppressor gene. Previous work from our lab has focused on the phenotypic characterisation of this mutant, establishing the links between vhl mutation, the hypoxic response and cancer. To further develop fish as a model for studying hypoxic signalling, we examine the transcriptional profile of the vhl mutant with respect to Hif-1α. As our approach uses embryos consisting of many cell types, it has the potential to uncover additional HIF regulated genes that have escaped detection in analogous mammalian cell culture studies.

Results: We performed high-density oligonucleotide microarray analysis of the gene expression changes in von Hippel-Lindau mutant zebrafish, which identified up-regulation of well-known hypoxia response genes and down-regulation of genes primarily involved in lipid processing. To identify the dependency of these transcriptional changes on HIF, we undertook Chromatin Immunoprecipitation linked next generation sequencing (ChIP-seq) for the transcription factor Hypoxia Inducible Factor 1α (HIF-1α). We identified HIF-1α binding sites across the genome, with binding sites showing enrichment for an RCGTG motif, showing conservation with the mammalian hypoxia response element.

Conclusions: Transcriptome analysis of vhl mutant embryos detected activation of key hypoxia response genes seen in human cell models of hypoxia, but also suppression of many genes primarily involved in lipid processing. ChIP-seq analysis of Hif-1α binding sites unveiled an unprecedented number of loci, with a high proportion containing a canonical hypoxia response element. Whether these sites are functional remains unknown, nevertheless their frequent location near transcriptional start sites suggests functionality, and will allow for investigation into the potential hypoxic regulation of genes in their vicinity. We expect that our data will be an excellent starting point for analysis of both fish and mammalian gene regulation by HIF.
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http://dx.doi.org/10.1186/s12864-015-2169-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642629PMC
November 2015

High-throughput and quantitative genome-wide messenger RNA sequencing for molecular phenotyping.

BMC Genomics 2015 Aug 5;16:578. Epub 2015 Aug 5.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridgeshire, CB10 1SA, UK.

Background: We present a genome-wide messenger RNA (mRNA) sequencing technique that converts small amounts of RNA from many samples into molecular phenotypes. It encompasses all steps from sample preparation to sequence analysis and is applicable to baseline profiling or perturbation measurements.

Results: Multiplex sequencing of transcript 3' ends identifies differential transcript abundance independent of gene annotation. We show that increasing biological replicate number while maintaining the total amount of sequencing identifies more differentially abundant transcripts.

Conclusions: This method can be implemented on polyadenylated RNA from any organism with an annotated reference genome and in any laboratory with access to Illumina sequencing.
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http://dx.doi.org/10.1186/s12864-015-1788-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4524448PMC
August 2015

Identification of a plant isoflavonoid that causes biliary atresia.

Sci Transl Med 2015 May;7(286):286ra67

Department of Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. Cell and Developmental Biology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.

Biliary atresia (BA) is a rapidly progressive and destructive fibrotic disorder of unknown etiology affecting the extrahepatic biliary tree of neonates. Epidemiological studies suggest that an environmental factor, such as a virus or toxin, is the cause of the disease, although none have been definitively established. Several naturally occurring outbreaks of BA in Australian livestock have been associated with the ingestion of unusual plants by pregnant animals during drought conditions. We used a biliary secretion assay in zebrafish to isolate a previously undescribed isoflavonoid, biliatresone, from Dysphania species implicated in a recent BA outbreak. This compound caused selective destruction of the extrahepatic, but not intrahepatic, biliary system of larval zebrafish. A mutation that enhanced biliatresone toxicity mapped to a region of the zebrafish genome that has conserved synteny with an established human BA susceptibility locus. The toxin also caused loss of cilia in neonatal mouse extrahepatic cholangiocytes in culture and disrupted cell polarity and monolayer integrity in cholangiocyte spheroids. Together, these findings provide direct evidence that BA could be initiated by perinatal exposure to an environmental toxin.
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http://dx.doi.org/10.1126/scitranslmed.aaa1652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784984PMC
May 2015

Clinical findings associated with homozygous sickle cell disease in the Barbadian population--do we need a national SCD registry?

BMC Res Notes 2014 Feb 22;7:102. Epub 2014 Feb 22.

Chronic Disease Research Centre, Tropical Medicine Research Institute, University of the West Indies, Jemmott's Lane, St, Michael, Brigetown, WI, Barbados.

Background: Comprehensive care in homozygous sickle cell disease (HbSS) entails universal neonatal screening and subsequent monitoring of identified patients, a process which has been streamlined in the neighbouring island of Jamaica. In preparation for a similar undertaking in Barbados, we have developed a database of persons with known HbSS, and have piloted processes for documenting clinical manifestations. We now present a brief clinical profile of these findings with comparisons to the Jamaican cohort.

Methods: HbSS participants were recruited from clinics and support groups. A history of select clinical symptoms was taken and blood and urine samples and echocardiograms were analysed. A re-analysis of data from a previous birth cohort was completed.

Results: Forty-eight persons participated (32 F/16 M); age range 10-62 yrs. 94% had a history of ever having a painful crisis. In the past year, 44% of participants had at least one crisis. There were >69 crises in 21 individuals; 61% were self-managed at home and the majority of the others were treated and discharged from hospital; few were admitted. The prevalence of chronic leg ulceration was 27%. Forty-two persons had urinalysis, 44% were diagnosed with albuminuria (urinary protein/creatinine ratio ≥30 mg/g). Thirty-two participants had echocardiography, 28% had a TRJV ≥ 2.5 m/s. Re-analysis of the incidence study revealed a sickle gene frequency (95% CI) of 2.01% (0.24 to 7.21).

Conclusion: Although we share a common ancestry, it is thought that HbSS is less common and less severe in Barbados compared to Jamaica. The Jamaican studies reported a sickle gene frequency of 3.15 (2.81 to 3.52); the prevalence of chronic leg ulcers and albuminuria was 29.5% and 42.5% respectively. These comparisons suggest that our initial thoughts may be speculative and that HbSS may be an underestimated clinical problem in Barbados. A prospective neonatal screening programme combined with centralized, routine monitoring of HbSS morbidity and outcomes will definitively answer this question and will improve the evidence-based care and management of HbSS in Barbados.
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http://dx.doi.org/10.1186/1756-0500-7-102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3936843PMC
February 2014

Identification of the zebrafish maternal and paternal transcriptomes.

Development 2013 Jul 29;140(13):2703-10. Epub 2013 May 29.

The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK.

Transcription is an essential component of basic cellular and developmental processes. However, early embryonic development occurs in the absence of transcription and instead relies upon maternal mRNAs and proteins deposited in the egg during oocyte maturation. Although the early zebrafish embryo is competent to transcribe exogenous DNA, factors present in the embryo maintain genomic DNA in a state that is incompatible with transcription. The cell cycles of the early embryo titrate out these factors, leading to zygotic transcription initiation, presumably in response to a change in genomic DNA chromatin structure to a state that supports transcription. To understand the molecular mechanisms controlling this maternal to zygotic transition, it is important to distinguish between the maternal and zygotic transcriptomes during this period. Here we use exome sequencing and RNA-seq to achieve such discrimination and in doing so have identified the first zygotic genes to be expressed in the embryo. Our work revealed different profiles of maternal mRNA post-transcriptional regulation prior to zygotic transcription initiation. Finally, we demonstrate that maternal mRNAs are required for different modes of zygotic transcription initiation, which is not simply dependent on the titration of factors that maintain genomic DNA in a transcriptionally incompetent state.
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http://dx.doi.org/10.1242/dev.095091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3678340PMC
July 2013

The zebrafish reference genome sequence and its relationship to the human genome.

Nature 2013 Apr 17;496(7446):498-503. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Zebrafish have become a popular organism for the study of vertebrate gene function. The virtually transparent embryos of this species, and the ability to accelerate genetic studies by gene knockdown or overexpression, have led to the widespread use of zebrafish in the detailed investigation of vertebrate gene function and increasingly, the study of human genetic disease. However, for effective modelling of human genetic disease it is important to understand the extent to which zebrafish genes and gene structures are related to orthologous human genes. To examine this, we generated a high-quality sequence assembly of the zebrafish genome, made up of an overlapping set of completely sequenced large-insert clones that were ordered and oriented using a high-resolution high-density meiotic map. Detailed automatic and manual annotation provides evidence of more than 26,000 protein-coding genes, the largest gene set of any vertebrate so far sequenced. Comparison to the human reference genome shows that approximately 70% of human genes have at least one obvious zebrafish orthologue. In addition, the high quality of this genome assembly provides a clearer understanding of key genomic features such as a unique repeat content, a scarcity of pseudogenes, an enrichment of zebrafish-specific genes on chromosome 4 and chromosomal regions that influence sex determination.
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http://dx.doi.org/10.1038/nature12111DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3703927PMC
April 2013

A systematic genome-wide analysis of zebrafish protein-coding gene function.

Nature 2013 Apr 17;496(7446):494-7. Epub 2013 Apr 17.

Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK.

Since the publication of the human reference genome, the identities of specific genes associated with human diseases are being discovered at a rapid rate. A central problem is that the biological activity of these genes is often unclear. Detailed investigations in model vertebrate organisms, typically mice, have been essential for understanding the activities of many orthologues of these disease-associated genes. Although gene-targeting approaches and phenotype analysis have led to a detailed understanding of nearly 6,000 protein-coding genes, this number falls considerably short of the more than 22,000 mouse protein-coding genes. Similarly, in zebrafish genetics, one-by-one gene studies using positional cloning, insertional mutagenesis, antisense morpholino oligonucleotides, targeted re-sequencing, and zinc finger and TAL endonucleases have made substantial contributions to our understanding of the biological activity of vertebrate genes, but again the number of genes studied falls well short of the more than 26,000 zebrafish protein-coding genes. Importantly, for both mice and zebrafish, none of these strategies are particularly suited to the rapid generation of knockouts in thousands of genes and the assessment of their biological activity. Here we describe an active project that aims to identify and phenotype the disruptive mutations in every zebrafish protein-coding gene, using a well-annotated zebrafish reference genome sequence, high-throughput sequencing and efficient chemical mutagenesis. So far we have identified potentially disruptive mutations in more than 38% of all known zebrafish protein-coding genes. We have developed a multi-allelic phenotyping scheme to efficiently assess the effects of each allele during embryogenesis and have analysed the phenotypic consequences of over 1,000 alleles. All mutant alleles and data are available to the community and our phenotyping scheme is adaptable to phenotypic analysis beyond embryogenesis.
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http://dx.doi.org/10.1038/nature11992DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3743023PMC
April 2013

Genomic regulatory blocks encompass multiple neighboring genes and maintain conserved synteny in vertebrates.

Genome Res 2007 May 26;17(5):545-55. Epub 2007 Mar 26.

Sars Centre for Marine Molecular Biology, University of Bergen, 5008 Bergen, Norway.

We report evidence for a mechanism for the maintenance of long-range conserved synteny across vertebrate genomes. We found the largest mammal-teleost conserved chromosomal segments to be spanned by highly conserved noncoding elements (HCNEs), their developmental regulatory target genes, and phylogenetically and functionally unrelated "bystander" genes. Bystander genes are not specifically under the control of the regulatory elements that drive the target genes and are expressed in patterns that are different from those of the target genes. Reporter insertions distal to zebrafish developmental regulatory genes pax6.1/2, rx3, id1, and fgf8 and miRNA genes mirn9-1 and mirn9-5 recapitulate the expression patterns of these genes even if located inside or beyond bystander genes, suggesting that the regulatory domain of a developmental regulatory gene can extend into and beyond adjacent transcriptional units. We termed these chromosomal segments genomic regulatory blocks (GRBs). After whole genome duplication in teleosts, GRBs, including HCNEs and target genes, were often maintained in both copies, while bystander genes were typically lost from one GRB, strongly suggesting that evolutionary pressure acts to keep the single-copy GRBs of higher vertebrates intact. We show that loss of bystander genes and other mutational events suffered by duplicated GRBs in teleost genomes permits target gene identification and HCNE/target gene assignment. These findings explain the absence of evolutionary breakpoints from large vertebrate chromosomal segments and will aid in the recognition of position effect mutations within human GRBs.
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http://dx.doi.org/10.1101/gr.6086307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1855176PMC
May 2007
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