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1.  Comparative Analysis of piggyBac, CRISPR/Cas9 and TALEN Mediated BAC Transgenesis in the Zygote for the Generation of Humanized SIRPA Rats 
Scientific Reports  2016;6:31455.
BAC transgenic mammalian systems offer an important platform for recapitulating human gene expression and disease modeling. While the larger body mass, and greater genetic and physiologic similarity to humans render rats well suited for reproducing human immune diseases and evaluating therapeutic strategies, difficulties of generating BAC transgenic rats have hindered progress. Thus, an efficient method for BAC transgenesis in rats would be valuable. Immunodeficient mice carrying a human SIRPA transgene have previously been shown to support improved human cell hematopoiesis. Here, we have generated for the first time, human SIRPA BAC transgenic rats, for which the gene is faithfully expressed, functionally active, and germline transmissible. To do this, human SIRPA BAC was modified with elements to work in coordination with genome engineering technologies-piggyBac, CRISPR/Cas9 or TALEN. Our findings show that piggyBac transposition is a more efficient approach than the classical BAC transgenesis, resulting in complete BAC integration with predictable end sequences, thereby permitting precise assessment of the integration site. Neither CRISPR/Cas9 nor TALEN increased BAC transgenesis. Therefore, an efficient generation of human SIRPA transgenic rats using piggyBac opens opportunities for expansion of humanized transgenic rat models in the future to advance biomedical research and therapeutic applications.
doi:10.1038/srep31455
PMCID: PMC4987655  PMID: 27530248
2.  Transcriptome Analysis of Targeted Mouse Mutations Reveals the Topography of Local Changes in Gene Expression 
PLoS Genetics  2016;12(2):e1005691.
The unintended consequences of gene targeting in mouse models have not been thoroughly studied and a more systematic analysis is needed to understand the frequency and characteristics of off-target effects. Using RNA-seq, we evaluated targeted and neighboring gene expression in tissues from 44 homozygous mutants compared with C57BL/6N control mice. Two allele types were evaluated: 15 targeted trap mutations (TRAP); and 29 deletion alleles (DEL), usually a deletion between the translational start and the 3’ UTR. Both targeting strategies insert a bacterial beta-galactosidase reporter (LacZ) and a neomycin resistance selection cassette. Evaluating transcription of genes in +/- 500 kb of flanking DNA around the targeted gene, we found up-regulated genes more frequently around DEL compared with TRAP alleles, however the frequency of alleles with local down-regulated genes flanking DEL and TRAP targets was similar. Down-regulated genes around both DEL and TRAP targets were found at a higher frequency than expected from a genome-wide survey. However, only around DEL targets were up-regulated genes found with a significantly higher frequency compared with genome-wide sampling. Transcriptome analysis confirms targeting in 97% of DEL alleles, but in only 47% of TRAP alleles probably due to non-functional splice variants, and some splicing around the gene trap. Local effects on gene expression are likely due to a number of factors including compensatory regulation, loss or disruption of intragenic regulatory elements, the exogenous promoter in the neo selection cassette, removal of insulating DNA in the DEL mutants, and local silencing due to disruption of normal chromatin organization or presence of exogenous DNA. An understanding of local position effects is important for understanding and interpreting any phenotype attributed to targeted gene mutations, or to spontaneous indels.
Author Summary
Insertion of foreign DNA into mammalian genomes, and the deletion of DNA, may have unintended consequences extending beyond the site of the mutation. In the mouse, the insertion of foreign DNA, including foreign regulatory DNA, combined with the deletion of part of the targeted gene, had striking effects on the regulation of neighboring genes. And this ectopic local gene dysregulation occurred at high frequency in regions of high gene density. These findings emphasize the importance of evaluating the local effects on gene regulation following spontaneous insertions and deletions, and after engineering mutations, in mammalian systems. Phenotypes associated with mutations in a specific gene may be partially or entirely due to effects on neighboring genes.
doi:10.1371/journal.pgen.1005691
PMCID: PMC4739719  PMID: 26839965
3.  Gibbon genome and the fast karyotype evolution of small apes 
Carbone, Lucia | Harris, R. Alan | Gnerre, Sante | Veeramah, Krishna R. | Lorente-Galdos, Belen | Huddleston, John | Meyer, Thomas J. | Herrero, Javier | Roos, Christian | Aken, Bronwen | Anaclerio, Fabio | Archidiacono, Nicoletta | Baker, Carl | Barrell, Daniel | Batzer, Mark A. | Beal, Kathryn | Blancher, Antoine | Bohrson, Craig L. | Brameier, Markus | Campbell, Michael S. | Capozzi, Oronzo | Casola, Claudio | Chiatante, Giorgia | Cree, Andrew | Damert, Annette | de Jong, Pieter J. | Dumas, Laura | Fernandez-Callejo, Marcos | Flicek, Paul | Fuchs, Nina V. | Gut, Marta | Gut, Ivo | Hahn, Matthew W. | Hernandez-Rodriguez, Jessica | Hillier, LaDeana W. | Hubley, Robert | Ianc, Bianca | Izsvák, Zsuzsanna | Jablonski, Nina G. | Johnstone, Laurel M. | Karimpour-Fard, Anis | Konkel, Miriam K. | Kostka, Dennis | Lazar, Nathan H. | Lee, Sandra L. | Lewis, Lora R. | Liu, Yue | Locke, Devin P. | Mallick, Swapan | Mendez, Fernando L. | Muffato, Matthieu | Nazareth, Lynne V. | Nevonen, Kimberly A. | O,Bleness, Majesta | Ochis, Cornelia | Odom, Duncan T. | Pollard, Katherine S. | Quilez, Javier | Reich, David | Rocchi, Mariano | Schumann, Gerald G. | Searle, Stephen | Sikela, James M. | Skollar, Gabriella | Smit, Arian | Sonmez, Kemal | Hallers, Boudewijn ten | Terhune, Elizabeth | Thomas, Gregg W.C. | Ullmer, Brygg | Ventura, Mario | Walker, Jerilyn A. | Wall, Jeffrey D. | Walter, Lutz | Ward, Michelle C. | Wheelan, Sarah J. | Whelan, Christopher W. | White, Simon | Wilhelm, Larry J. | Woerner, August E. | Yandell, Mark | Zhu, Baoli | Hammer, Michael F. | Marques-Bonet, Tomas | Eichler, Evan E. | Fulton, Lucinda | Fronick, Catrina | Muzny, Donna M. | Warren, Wesley C. | Worley, Kim C. | Rogers, Jeffrey | Wilson, Richard K. | Gibbs, Richard A.
Nature  2014;513(7517):195-201.
Gibbons are small arboreal apes that display an accelerated rate of evolutionary chromosomal rearrangement and occupy a key node in the primate phylogeny between Old World monkeys and great apes. Here we present the assembly and analysis of a northern white-cheeked gibbon (Nomascus leucogenys) genome. We describe the propensity for a gibbon-specific retrotransposon (LAVA) to insert into chromosome segregation genes and alter transcription by providing a premature termination site, suggesting a possible molecular mechanism for the genome plasticity of the gibbon lineage. We further show that the gibbon genera (Nomascus, Hylobates, Hoolock and Symphalangus) experienced a near-instantaneous radiation ~5 million years ago, coincident with major geographical changes in Southeast Asia that caused cycles of habitat compression and expansion. Finally, we identify signatures of positive selection in genes important for forelimb development (TBX5) and connective tissues (COL1A1) that may have been involved in the adaptation of gibbons to their arboreal habitat.
doi:10.1038/nature13679
PMCID: PMC4249732  PMID: 25209798
4.  Sphingosine-1-phosphate lyase downregulation promotes colon carcinogenesis through STAT3-activated microRNAs 
The Journal of Clinical Investigation  2014;124(12):5368-5384.
Growing evidence supports a link between inflammation and cancer; however, mediators of the transition between inflammation and carcinogenesis remain incompletely understood. Sphingosine-1-phosphate (S1P) lyase (SPL) irreversibly degrades the bioactive sphingolipid S1P and is highly expressed in enterocytes but downregulated in colon cancer. Here, we investigated the role of SPL in colitis-associated cancer (CAC). We generated mice with intestinal epithelium-specific Sgpl1 deletion and chemically induced colitis and tumor formation in these animals. Compared with control animals, mice lacking intestinal SPL exhibited greater disease activity, colon shortening, cytokine levels, S1P accumulation, tumors, STAT3 activation, STAT3-activated microRNAs (miRNAs), and suppression of miR-targeted anti-oncogene products. This phenotype was attenuated by STAT3 inhibition. In fibroblasts, silencing SPL promoted tumorigenic transformation through a pathway involving extracellular transport of S1P through S1P transporter spinster homolog 2 (SPNS2), S1P receptor activation, JAK2/STAT3-dependent miR-181b-1 induction, and silencing of miR-181b-1 target cylindromatosis (CYLD). Colon biopsies from patients with inflammatory bowel disease revealed enhanced S1P and STAT3 signaling. In mice with chemical-induced CAC, oral administration of plant-type sphingolipids called sphingadienes increased colonic SPL levels and reduced S1P levels, STAT3 signaling, cytokine levels, and tumorigenesis, indicating that SPL prevents transformation and carcinogenesis. Together, our results suggest that dietary sphingolipids can augment or prevent colon cancer, depending upon whether they are metabolized to S1P or promote S1P metabolism through the actions of SPL.
doi:10.1172/JCI74188
PMCID: PMC4348973  PMID: 25347472
5.  Sequencing and Assembly of the 22-Gb Loblolly Pine Genome 
Genetics  2014;196(3):875-890.
Conifers are the predominant gymnosperm. The size and complexity of their genomes has presented formidable technical challenges for whole-genome shotgun sequencing and assembly. We employed novel strategies that allowed us to determine the loblolly pine (Pinus taeda) reference genome sequence, the largest genome assembled to date. Most of the sequence data were derived from whole-genome shotgun sequencing of a single megagametophyte, the haploid tissue of a single pine seed. Although that constrained the quantity of available DNA, the resulting haploid sequence data were well-suited for assembly. The haploid sequence was augmented with multiple linking long-fragment mate pair libraries from the parental diploid DNA. For the longest fragments, we used novel fosmid DiTag libraries. Sequences from the linking libraries that did not match the megagametophyte were identified and removed. Assembly of the sequence data were aided by condensing the enormous number of paired-end reads into a much smaller set of longer “super-reads,” rendering subsequent assembly with an overlap-based assembly algorithm computationally feasible. To further improve the contiguity and biological utility of the genome sequence, additional scaffolding methods utilizing independent genome and transcriptome assemblies were implemented. The combination of these strategies resulted in a draft genome sequence of 20.15 billion bases, with an N50 scaffold size of 66.9 kbp.
doi:10.1534/genetics.113.159715
PMCID: PMC3948813  PMID: 24653210
placeholder; loblolly pine; (see cocoa genome); Pinus taeda L.; conifer; genome; de novo assembly; fosmid
6.  Unique Features of the Loblolly Pine (Pinus taeda L.) Megagenome Revealed Through Sequence Annotation 
Genetics  2014;196(3):891-909.
The largest genus in the conifer family Pinaceae is Pinus, with over 100 species. The size and complexity of their genomes (∼20–40 Gb, 2n = 24) have delayed the arrival of a well-annotated reference sequence. In this study, we present the annotation of the first whole-genome shotgun assembly of loblolly pine (Pinus taeda L.), which comprises 20.1 Gb of sequence. The MAKER-P annotation pipeline combined evidence-based alignments and ab initio predictions to generate 50,172 gene models, of which 15,653 are classified as high confidence. Clustering these gene models with 13 other plant species resulted in 20,646 gene families, of which 1554 are predicted to be unique to conifers. Among the conifer gene families, 159 are composed exclusively of loblolly pine members. The gene models for loblolly pine have the highest median and mean intron lengths of 24 fully sequenced plant genomes. Conifer genomes are full of repetitive DNA, with the most significant contributions from long-terminal-repeat retrotransposons. In depth analysis of the tandem and interspersed repetitive content yielded a combined estimate of 82%.
doi:10.1534/genetics.113.159996
PMCID: PMC3948814  PMID: 24653211
introns; gene family; repeats; retrotransposons; conifer
7.  The Common Marmoset Genome Provides Insight into Primate Biology and Evolution 
Worley, Kim C. | Warren, Wesley C. | Rogers, Jeffrey | Locke, Devin | Muzny, Donna M. | Mardis, Elaine R. | Weinstock, George M. | Tardif, Suzette D. | Aagaard, Kjersti M. | Archidiacono, Nicoletta | Rayan, Nirmala Arul | Batzer, Mark A. | Beal, Kathryn | Brejova, Brona | Capozzi, Oronzo | Capuano, Saverio B. | Casola, Claudio | Chandrabose, Mimi M. | Cree, Andrew | Dao, Marvin Diep | de Jong, Pieter J. | del Rosario, Ricardo Cruz-Herrera | Delehaunty, Kim D. | Dinh, Huyen H. | Eichler, Evan | Fitzgerald, Stephen | Flicek, Paul | Fontenot, Catherine C. | Fowler, R. Gerald | Fronick, Catrina | Fulton, Lucinda A. | Fulton, Robert S. | Gabisi, Ramatu Ayiesha | Gerlach, Daniel | Graves, Tina A. | Gunaratne, Preethi H. | Hahn, Matthew W. | Haig, David | Han, Yi | Harris, R. Alan | Herrero, Javier M. | Hillier, LaDeana W. | Hubley, Robert | Hughes, Jennifer F. | Hume, Jennifer | Jhangiani, Shalini N. | Jorde, Lynn B. | Joshi, Vandita | Karakor, Emre | Konkel, Miriam K. | Kosiol, Carolin | Kovar, Christie L. | Kriventseva, Evgenia V. | Lee, Sandra L. | Lewis, Lora R. | Liu, Yih-shin | Lopez, John | Lopez-Otin, Carlos | Lorente-Galdos, Belen | Mansfield, Keith G. | Marques-Bonet, Tomas | Minx, Patrick | Misceo, Doriana | Moncrieff, J. Scott | Morgan, Margaret B. | Muthuswamy, Raveendran | Nazareth, Lynne V. | Newsham, Irene | Nguyen, Ngoc Bich | Okwuonu, Geoffrey O. | Prabhakar, Shyam | Perales, Lora | Pu, Ling-Ling | Puente, Xose S. | Quesada, Victor | Ranck, Megan C. | Raney, Brian J. | Deiros, David Rio | Rocchi, Mariano | Rodriguez, David | Ross, Corinna | Ruffier, Magali | Ruiz, San Juana | Sajjadian, S. | Santibanez, Jireh | Schrider, Daniel R. | Searle, Steve | Skaletsky, Helen | Soibam, Benjamin | Smit, Arian F. A. | Tennakoon, Jayantha B. | Tomaska, Lubomir | Ullmer, Brygg | Vejnar, Charles E. | Ventura, Mario | Vilella, Albert J. | Vinar, Tomas | Vogel, Jan-Hinnerk | Walker, Jerilyn A. | Wang, Qing | Warner, Crystal M. | Wildman, Derek E. | Witherspoon, David J. | Wright, Rita A. | Wu, Yuanqing | Xiao, Weimin | Xing, Jinchuan | Zdobnov, Evgeny M. | Zhu, Baoli | Gibbs, Richard A. | Wilson, Richard K.
Nature genetics  2014;46(8):850-857.
A first analysis of the genome sequence of the common marmoset (Callithrix jacchus), assembled using traditional Sanger methods and Ensembl annotation, has permitted genomic comparison with apes and that old world monkeys and the identification of specific molecular features a rapid reproductive capacity partly due to may contribute to the unique biology of diminutive The common marmoset has prevalence of this dizygotic primate. twins. Remarkably, these twins share placental circulation and exchange hematopoietic stem cells in utero, resulting in adults that are hematopoietic chimeras.
We observed positive selection or non-synonymous substitutions for genes encoding growth hormone / insulin-like growth factor (growth pathways), respiratory complex I (metabolic pathways), immunobiology, and proteases (reproductive and immunity pathways). In addition, both protein-coding and microRNA genes related to reproduction exhibit rapid sequence evolution. This New World monkey genome sequence enables significantly increased power for comparative analyses among available primate genomes and facilitates biomedical research application.
doi:10.1038/ng.3042
PMCID: PMC4138798  PMID: 25038751
8.  Reciprocal knock-in mice to investigate the functional redundancy of lamin B1 and lamin B2 
Molecular Biology of the Cell  2014;25(10):1666-1675.
To assess the redundancy of lamins B1 and B2, knock-in lines were created that produce lamin B2 from the Lmnb1 locus and lamin B1 from the Lmnb2 locus. Both lines developed severe neurodevelopmental abnormalities, indicating that the abnormalities elicited by the loss of one B-type lamin cannot be prevented by increased synthesis of the other.
Lamins B1 and B2 (B-type lamins) have very similar sequences and are expressed ubiquitously. In addition, both Lmnb1- and Lmnb2-deficient mice die soon after birth with neuronal layering abnormalities in the cerebral cortex, a consequence of defective neuronal migration. The similarities in amino acid sequences, expression patterns, and knockout phenotypes raise the question of whether the two proteins have redundant functions. To investigate this topic, we generated “reciprocal knock-in mice”—mice that make lamin B2 from the Lmnb1 locus (Lmnb1B2/B2) and mice that make lamin B1 from the Lmnb2 locus (Lmnb2B1/B1). Lmnb1B2/B2 mice produced increased amounts of lamin B2 but no lamin B1; they died soon after birth with neuronal layering abnormalities in the cerebral cortex. However, the defects in Lmnb1B2/B2 mice were less severe than those in Lmnb1-knockout mice, indicating that increased amounts of lamin B2 partially ameliorate the abnormalities associated with lamin B1 deficiency. Similarly, increased amounts of lamin B1 in Lmnb2B1/B1 mice did not prevent the neurodevelopmental defects elicited by lamin B2 deficiency. We conclude that lamins B1 and B2 have unique roles in the developing brain and that increased production of one B-type lamin does not fully complement loss of the other.
doi:10.1091/mbc.E14-01-0683
PMCID: PMC4019497  PMID: 24672053
9.  The zebrafish reference genome sequence and its relationship to the human genome 
Howe, Kerstin | Clark, Matthew D. | Torroja, Carlos F. | Torrance, James | Berthelot, Camille | Muffato, Matthieu | Collins, John E. | Humphray, Sean | McLaren, Karen | Matthews, Lucy | McLaren, Stuart | Sealy, Ian | Caccamo, Mario | Churcher, Carol | Scott, Carol | Barrett, Jeffrey C. | Koch, Romke | Rauch, Gerd-Jörg | White, Simon | Chow, William | Kilian, Britt | Quintais, Leonor T. | Guerra-Assunção, José A. | Zhou, Yi | Gu, Yong | Yen, Jennifer | Vogel, Jan-Hinnerk | Eyre, Tina | Redmond, Seth | Banerjee, Ruby | Chi, Jianxiang | Fu, Beiyuan | Langley, Elizabeth | Maguire, Sean F. | Laird, Gavin K. | Lloyd, David | Kenyon, Emma | Donaldson, Sarah | Sehra, Harminder | Almeida-King, Jeff | Loveland, Jane | Trevanion, Stephen | Jones, Matt | Quail, Mike | Willey, Dave | Hunt, Adrienne | Burton, John | Sims, Sarah | McLay, Kirsten | Plumb, Bob | Davis, Joy | Clee, Chris | Oliver, Karen | Clark, Richard | Riddle, Clare | Eliott, David | Threadgold, Glen | Harden, Glenn | Ware, Darren | Mortimer, Beverly | Kerry, Giselle | Heath, Paul | Phillimore, Benjamin | Tracey, Alan | Corby, Nicole | Dunn, Matthew | Johnson, Christopher | Wood, Jonathan | Clark, Susan | Pelan, Sarah | Griffiths, Guy | Smith, Michelle | Glithero, Rebecca | Howden, Philip | Barker, Nicholas | Stevens, Christopher | Harley, Joanna | Holt, Karen | Panagiotidis, Georgios | Lovell, Jamieson | Beasley, Helen | Henderson, Carl | Gordon, Daria | Auger, Katherine | Wright, Deborah | Collins, Joanna | Raisen, Claire | Dyer, Lauren | Leung, Kenric | Robertson, Lauren | Ambridge, Kirsty | Leongamornlert, Daniel | McGuire, Sarah | Gilderthorp, Ruth | Griffiths, Coline | Manthravadi, Deepa | Nichol, Sarah | Barker, Gary | Whitehead, Siobhan | Kay, Michael | Brown, Jacqueline | Murnane, Clare | Gray, Emma | Humphries, Matthew | Sycamore, Neil | Barker, Darren | Saunders, David | Wallis, Justene | Babbage, Anne | Hammond, Sian | Mashreghi-Mohammadi, Maryam | Barr, Lucy | Martin, Sancha | Wray, Paul | Ellington, Andrew | Matthews, Nicholas | Ellwood, Matthew | Woodmansey, Rebecca | Clark, Graham | Cooper, James | Tromans, Anthony | Grafham, Darren | Skuce, Carl | Pandian, Richard | Andrews, Robert | Harrison, Elliot | Kimberley, Andrew | Garnett, Jane | Fosker, Nigel | Hall, Rebekah | Garner, Patrick | Kelly, Daniel | Bird, Christine | Palmer, Sophie | Gehring, Ines | Berger, Andrea | Dooley, Christopher M. | Ersan-Ürün, Zübeyde | Eser, Cigdem | Geiger, Horst | Geisler, Maria | Karotki, Lena | Kirn, Anette | Konantz, Judith | Konantz, Martina | Oberländer, Martina | Rudolph-Geiger, Silke | Teucke, Mathias | Osoegawa, Kazutoyo | Zhu, Baoli | Rapp, Amanda | Widaa, Sara | Langford, Cordelia | Yang, Fengtang | Carter, Nigel P. | Harrow, Jennifer | Ning, Zemin | Herrero, Javier | Searle, Steve M. J. | Enright, Anton | Geisler, Robert | Plasterk, Ronald H. A. | Lee, Charles | Westerfield, Monte | de Jong, Pieter J. | Zon, Leonard I. | Postlethwait, John H. | Nüsslein-Volhard, Christiane | Hubbard, Tim J. P. | Crollius, Hugues Roest | Rogers, Jane | Stemple, Derek L.
Nature  2013;496(7446):498-503.
Zebrafish have become a popular organism for the study of vertebrate gene function1,2. 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 disease3–5. 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 genes6, 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.
doi:10.1038/nature12111
PMCID: PMC3703927  PMID: 23594743
10.  microRNA-1 regulates sarcomere formation and suppresses smooth muscle gene expression in the mammalian heart 
eLife  2013;2:e01323.
microRNA-1 (miR-1) is an evolutionarily conserved, striated muscle-enriched miRNA. Most mammalian genomes contain two copies of miR-1, and in mice, deletion of a single locus, miR-1-2, causes incompletely penetrant lethality and subtle cardiac defects. Here, we report that deletion of miR-1-1 resulted in a phenotype similar to that of the miR-1-2 mutant. Compound miR-1 knockout mice died uniformly before weaning due to severe cardiac dysfunction. miR-1-null cardiomyocytes had abnormal sarcomere organization and decreased phosphorylation of the regulatory myosin light chain-2 (MLC2), a critical cytoskeletal regulator. The smooth muscle-restricted inhibitor of MLC2 phosphorylation, Telokin, was ectopically expressed in the myocardium, along with other smooth muscle genes. miR-1 repressed Telokin expression through direct targeting and by repressing its transcriptional regulator, Myocardin. Our results reveal that miR-1 is required for postnatal cardiac function and reinforces the striated muscle phenotype by regulating both transcriptional and effector nodes of the smooth muscle gene expression network.
DOI: http://dx.doi.org/10.7554/eLife.01323.001
eLife digest
MicroRNAs are tiny RNAs that do not encode proteins. Instead, they regulate the expression of genes by preventing protein-encoding messenger RNAs from being translated into protein. MicroRNAs are expressed throughout the body, including the heart, where the most abundant microRNA is called miR-1. This is encoded by two nearly identical genes: miR-1-1 and miR-1-2.
Mice that lack the miR-1-2 gene have various heart abnormalities, but generally survive because they still produce some miR-1 from their remaining miR-1-1 gene. Now, Heidersbach et al. have generated the first mice that specifically lack both miR-1 genes, and shown that these animals die before weaning.
When viewed under the electron microscope, heart muscle from miR-1 double knockout mice lacks the characteristic ‘striped’, or striated, appearance of normal heart muscle. Additionally, miR-1 double knockout hearts have some gene expression characteristics more similar to the smooth muscle found in the gut and in the walls of blood vessels. Smooth muscle differs from striated muscle in that it lacks sarcomeres: these are bands of fibrous proteins, such as myosin, that are essential for muscle contraction.
In normal mice, an enzyme called MLCK contributes to the formation and function of sarcomeres by adding phosphate groups to myosin molecules. By contrast, in smooth muscle an enzyme called Telokin promotes phosphate group removal, and thus affects the function of sarcomeres. Heidersbach et al. showed that miR-1 interacts directly with Telokin mRNA to prevent its expression in the heart, and simultaneously represses a protein called Myocardin, which directly activates transcription of Telokin. However, when miR-1 is absent, as in the miR-1 double knockout mice, Telokin is expressed in heart muscle, along with many other genes characteristic of smooth muscle.
As well as improving our understanding of the development and functioning of the heart, these findings should shed new light on the role of microRNAs in maintaining the patterns of gene expression that characterize unique cell fates.
DOI: http://dx.doi.org/10.7554/eLife.01323.002
doi:10.7554/eLife.01323
PMCID: PMC3833424  PMID: 24252873
microRNA-1; cardiac; sarcomere; Telokin; Myocardin; smooth muscle gene expression; Mouse
11.  An Alu-Based Phylogeny of Gibbons (Hylobatidae) 
Molecular Biology and Evolution  2012;29(11):3441-3450.
Gibbons (Hylobatidae) are small, arboreal apes indigenous to Southeast Asia that diverged from other apes ∼15–18 Ma. Extant lineages radiated rapidly 6–10 Ma and are organized into four genera (Hylobates, Hoolock, Symphalangus, and Nomascus) consisting of 12–19 species. The use of short interspersed elements (SINEs) as phylogenetic markers has seen recent popularity due to several desirable characteristics: the ancestral state of a locus is known to be the absence of an element, rare potentially homoplasious events are relatively easy to resolve, and samples can be quickly and inexpensively genotyped. During radiation of primates, one particular family of SINEs, the Alu family, has proliferated in primate genomes. Nomascus leucogenys (northern white-cheeked gibbon) sequences were analyzed for repetitive content with RepeatMasker using a custom library. The sequences containing Alu elements identified as members of a gibbon-specific subfamily were then compared with orthologous positions in other primate genomes. A primate phylogenetic panel consisting of 18 primate species, including 13 gibbon species representing all four extant genera, was assayed for all loci, and a total of 125 gibbon-specific Alu insertions were identified. The resulting amplification patterns were used to generate a phylogenetic tree. We demonstrate significant support for Symphalangus as the most basal lineage within the family. Our findings also place Nomascus as a derived lineage, sister to Hoolock, with the Nomascus–Hoolock clade sister to Hylobates. Further, our analysis groups N. leucogenys and Nomascus siki as sister taxa to the exclusion of the other Nomascus species assayed. This study represents the first use of SINEs to determine the genus level phylogenetic relationships within the family Hylobatidae. These relationships have been resolved with robust support at most internal nodes, demonstrating the utility of SINE-based phylogenetic analysis. We postulate that hybridization and rapid radiation may have contributed to the complex and contradictory findings of the previous studies. Our findings will aid in the conservation of these threatened primates and inform future studies of the biogeographical history and distribution of modern gibbon species.
doi:10.1093/molbev/mss149
PMCID: PMC3472497  PMID: 22683814
primate; ape; phylogeny; retrotransposon; SINE; mobile elements
12.  Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution 
Nature genetics  2013;45(4):415-421e2.
Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species. Analyses of the assembly indicate that two whole-genome duplications likely occurred before the divergence of ancestral lamprey and gnathostome lineages. Moreover, the results help define key evolutionary events within vertebrate lineages, including the origin of myelin-associated proteins and the development of appendages. The lamprey genome provides an important resource for reconstructing vertebrate origins and the evolutionary events that have shaped the genomes of extant organisms.
doi:10.1038/ng.2568
PMCID: PMC3709584  PMID: 23435085
13.  Identification of novel candidate genes associated with cleft lip and palate using array comparative genomic hybridization 
Journal of medical genetics  2007;45(2):81-86.
Introduction and methods
We analyzed DNA samples isolated from individuals born with cleft lip and cleft palate to identify deletions and duplications of candidate gene loci using array comparative genomic hybridization (array-CGH).
Results
Of 83 syndromic cases analyzed we identified one subject with a previously unknown 2.7 Mb deletion at 22q11.21 coinciding with the DiGeorge syndrome region. Eighteen of the syndromic cases had clinical features of Van der Woude syndrome and deletions were identified in 5 of these, all of which encompassed the interferon regulatory factor 6 (IRF6) gene. In a series of 104 nonsyndromic cases we found one subject with a 3.2 Mb deletion at chromosome 6q25.1-25.2 and another with a 2.2 Mb deletion at 10q26.11-26.13. Analyses of parental DNA demonstrated that the two deletion cases at 22q11.21 and 6q25.1-25.2 were de novo, while the deletion of 10q26.11-26.13 was inherited from the mother, who also has cleft lip. These deletions appear likely to be causally associated with the phenotypes of the subjects. Estrogen receptor 1 (ESR1) and fibroblast growth factor receptor 2 (FGFR2) genes from the 6q25.1-25.2 and 10q26.11-26.13, respectively, were identified as likely causative genes using a gene prioritization software.
Discussion
We have shown that array-CGH analysis of DNA samples derived from cleft lip and palate subjects is an efficient and productive method for identifying candidate chromosomal loci and genes, complementing traditional genetic mapping strategies.
doi:10.1136/jmg.2007.052191
PMCID: PMC3732463  PMID: 17873121
cleft lip; cleft palate; array-CGH; candidate gene
14.  A conditional knockout resource for the genome–wide study of mouse gene function 
Nature  2011;474(7351):337-342.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
doi:10.1038/nature10163
PMCID: PMC3572410  PMID: 21677750
15.  Incomplete Lineage Sorting Is Common in Extant Gibbon Genera 
PLoS ONE  2013;8(1):e53682.
We sequenced reduced representation libraries by means of Illumina technology to generate over 1.5 Mb of orthologous sequence from a representative of each of the four extant gibbon genera (Nomascus, Hylobates, Symphalangus, and Hoolock). We used these data to assess the evolutionary relationships between the genera by evaluating the likelihoods of all possible bifurcating trees involving the four taxa. Our analyses provide weak support for a tree with Nomascus and Hylobates as sister taxa and with Hoolock and Symphalangus as sister taxa, though bootstrap resampling suggests that other phylogenetic scenarios are also possible. This uncertainty is due to short internal branch lengths and extensive incomplete lineage sorting across taxa. The true phylogenetic relationships among gibbon genera will likely require a more extensive whole-genome sequence analysis.
doi:10.1371/journal.pone.0053682
PMCID: PMC3544895  PMID: 23341974
16.  Human-specific evolution of novel SRGAP2 genes by incomplete segmental duplication 
Cell  2012;149(4):912-922.
SUMMARY
Gene duplication is an important source of phenotypic change and adaptive evolution. We use a novel genomic approach to identify highly identical sequence missing from the reference genome, confirming the cortical development gene Slit-Robo Rho GTPase activating protein 2 (SRGAP2) duplicated three times in humans. We show that the promoter and first nine exons of SRGAP2 duplicated from 1q32.1 (SRGAP2A) to 1q21.1 (SRGAP2B) ~3.4 million years ago (mya). Two larger duplications later copied SRGAP2B to chromosome 1p12 (SRGAP2C) and to proximal 1q21.1 (SRGAP2D), ~2.4 and ~1 mya, respectively. Sequence and expression analysis shows SRGAP2C is the most likely duplicate to encode a functional protein and among the most fixed human-specific duplicate genes. Our data suggest a mechanism where incomplete duplication created a novel function —at birth, antagonizing parental SRGAP2 function 2–3 mya a time corresponding to the transition from Australopithecus to Homo and the beginning of neocortex expansion.
doi:10.1016/j.cell.2012.03.033
PMCID: PMC3365555  PMID: 22559943
17.  The Genome of Chelonid Herpesvirus 5 Harbors Atypical Genes 
PLoS ONE  2012;7(10):e46623.
The Chelonid fibropapilloma-associated herpesvirus (CFPHV; ChHV5) is believed to be the causative agent of fibropapillomatosis (FP), a neoplastic disease of marine turtles. While clinical signs and pathology of FP are well known, research on ChHV5 has been impeded because no cell culture system for its propagation exists. We have cloned a BAC containing ChHV5 in pTARBAC2.1 and determined its nucleotide sequence. Accordingly, ChHV5 has a type D genome and its predominant gene order is typical for the varicellovirus genus within the alphaherpesvirinae. However, at least four genes that are atypical for an alphaherpesvirus genome were also detected, i.e. two members of the C-type lectin-like domain superfamily (F-lec1, F-lec2), an orthologue to the mouse cytomegalovirus M04 (F-M04) and a viral sialyltransferase (F-sial). Four lines of evidence suggest that these atypical genes are truly part of the ChHV5 genome: (1) the pTARBAC insertion interrupted the UL52 ORF, leaving parts of the gene to either side of the insertion and suggesting that an intact molecule had been cloned. (2) Using FP-associated UL52 (F-UL52) as an anchor and the BAC-derived sequences as a means to generate primers, overlapping PCR was performed with tumor-derived DNA as template, which confirmed the presence of the same stretch of “atypical” DNA in independent FP cases. (3) Pyrosequencing of DNA from independent tumors did not reveal previously undetected viral sequences, suggesting that no apparent loss of viral sequence had happened due to the cloning strategy. (4) The simultaneous presence of previously known ChHV5 sequences and F-sial as well as F-M04 sequences was also confirmed in geographically distinct Australian cases of FP. Finally, transcripts of F-sial and F-M04 but not transcripts of lytic viral genes were detected in tumors from Hawaiian FP-cases. Therefore, we suggest that F-sial and F-M04 may play a role in FP pathogenesis.
doi:10.1371/journal.pone.0046623
PMCID: PMC3462797  PMID: 23056373
18.  An absence of both lamin B1 and lamin B2 in keratinocytes has no effect on cell proliferation or the development of skin and hair 
Human Molecular Genetics  2011;20(18):3537-3544.
Nuclear lamins are usually classified as A-type (lamins A and C) or B-type (lamins B1 and B2). A-type lamins have been implicated in multiple genetic diseases but are not required for cell growth or development. In contrast, B-type lamins have been considered essential in eukaryotic cells, with crucial roles in DNA replication and in the formation of the mitotic spindle. Knocking down the genes for B-type lamins (LMNB1, LMNB2) in HeLa cells has been reported to cause apoptosis. In the current study, we created conditional knockout alleles for mouse Lmnb1 and Lmnb2, with the goal of testing the hypothesis that B-type lamins are crucial for the growth and viability of mammalian cells in vivo. Using the keratin 14-Cre transgene, we bred mice lacking the expression of both Lmnb1 and Lmnb2 in skin keratinocytes (Lmnb1Δ/ΔLmnb2Δ/Δ). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1Δ/ΔLmnb2Δ/Δ mice, and lamin B1 and lamin B2 proteins were undetectable. But despite an absence of B-type lamins in keratinocytes, the skin and hair of Lmnb1Δ/ΔLmnb2Δ/Δ mice developed normally and were free of histological abnormalities, even in 2-year-old mice. After an intraperitoneal injection of bromodeoxyuridine (BrdU), similar numbers of BrdU-positive keratinocytes were observed in the skin of wild-type and Lmnb1Δ/ΔLmnb2Δ/Δ mice. Lmnb1Δ/ΔLmnb2Δ/Δ keratinocytes did not exhibit aneuploidy, and their growth rate was normal in culture. These studies challenge the concept that B-type lamins are essential for proliferation and vitality of eukaryotic cells.
doi:10.1093/hmg/ddr266
PMCID: PMC3159554  PMID: 21659336
19.  The mammalian gene function resource: the international knockout mouse consortium 
Bradley, Allan | Anastassiadis, Konstantinos | Ayadi, Abdelkader | Battey, James F. | Bell, Cindy | Birling, Marie-Christine | Bottomley, Joanna | Brown, Steve D. | Bürger, Antje | Bult, Carol J. | Bushell, Wendy | Collins, Francis S. | Desaintes, Christian | Doe, Brendan | Economides, Aris | Eppig, Janan T. | Finnell, Richard H. | Fletcher, Colin | Fray, Martin | Frendewey, David | Friedel, Roland H. | Grosveld, Frank G. | Hansen, Jens | Hérault, Yann | Hicks, Geoffrey | Hörlein, Andreas | Houghton, Richard | Hrabé de Angelis, Martin | Huylebroeck, Danny | Iyer, Vivek | de Jong, Pieter J. | Kadin, James A. | Kaloff, Cornelia | Kennedy, Karen | Koutsourakis, Manousos | Kent Lloyd, K. C. | Marschall, Susan | Mason, Jeremy | McKerlie, Colin | McLeod, Michael P. | von Melchner, Harald | Moore, Mark | Mujica, Alejandro O. | Nagy, Andras | Nefedov, Mikhail | Nutter, Lauryl M. | Pavlovic, Guillaume | Peterson, Jane L. | Pollock, Jonathan | Ramirez-Solis, Ramiro | Rancourt, Derrick E. | Raspa, Marcello | Remacle, Jacques E. | Ringwald, Martin | Rosen, Barry | Rosenthal, Nadia | Rossant, Janet | Ruiz Noppinger, Patricia | Ryder, Ed | Schick, Joel Zupicich | Schnütgen, Frank | Schofield, Paul | Seisenberger, Claudia | Selloum, Mohammed | Simpson, Elizabeth M. | Skarnes, William C. | Smedley, Damian | Stanford, William L. | Francis Stewart, A. | Stone, Kevin | Swan, Kate | Tadepally, Hamsa | Teboul, Lydia | Tocchini-Valentini, Glauco P. | Valenzuela, David | West, Anthony P. | Yamamura, Ken-ichi | Yoshinaga, Yuko | Wurst, Wolfgang
Mammalian Genome  2012;23(9-10):580-586.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
doi:10.1007/s00335-012-9422-2
PMCID: PMC3463800  PMID: 22968824
20.  Long-Range Massively Parallel Mate Pair Sequencing Detects Distinct Mutations and Similar Patterns of Structural Mutability in Two Breast Cancer Cell Lines 
Cancer genetics  2011;204(8):447-457.
Cancer genomes frequently undergo genomic instability resulting in accumulation of chromosomal rearrangement. To date, one of the main challenges has been to confidently and accurately identify these rearrangements using short-read massively parallel sequencing. We were able to improve cancer rearrangement detection by combining two distinct massively parallel sequencing strategies: fosmid-sized (36 Kilobases on average) and standard 5 Kilobase mate pair libraries. We applied this strategy to map rearrangements in two breast cancer cell lines, MCF7 and HCC1954. We detect and validate a total of 91 somatic rearrangements in MCF7 and 25 in HCC1954, including genomic alterations corresponding to previously reported transcript aberrations in these two cell lines. Each of the genomes contains two types of breakpoints – clustered and dispersed. In both cell lines, the dispersed breakpoints show enrichment for low copy repeats, while the clustered breakpoints associate with high-copy number amplifications. Comparing the two genomes, we observe highly similar structural mutational spectra affecting different sets of genes, pointing to similar histories of genomic instability against the background of very different gene network perturbations.
doi:10.1016/j.cancergen.2011.07.009
PMCID: PMC3185296  PMID: 21962895
fosmid ditag; massively parallel sequencing; gene fusion; copy number variation; genomic instability
21.  Centromere Remodeling in Hoolock leuconedys (Hylobatidae) by a New Transposable Element Unique to the Gibbons 
Genome Biology and Evolution  2012;4(7):760-770.
Gibbons (Hylobatidae) shared a common ancestor with the other hominoids only 15–18 million years ago. Nevertheless, gibbons show very distinctive features that include heavily rearranged chromosomes. Previous observations indicate that this phenomenon may be linked to the attenuated epigenetic repression of transposable elements (TEs) in gibbon species. Here we describe the massive expansion of a repeat in almost all the centromeres of the eastern hoolock gibbon (Hoolock leuconedys). We discovered that this repeat is a new composite TE originating from the combination of portions of three other elements (L1ME5, AluSz6, and SVA_A) and thus named it LAVA. We determined that this repeat is found in all the gibbons but does not occur in other hominoids. Detailed investigation of 46 different LAVA elements revealed that the majority of them have target site duplications (TSDs) and a poly-A tail, suggesting that they have been retrotransposing in the gibbon genome. Although we did not find a direct correlation between the emergence of LAVA elements and human–gibbon synteny breakpoints, this new composite transposable element is another mark of the great plasticity of the gibbon genome. Moreover, the centromeric expansion of LAVA insertions in the hoolock closely resembles the massive centromeric expansion of the KERV-1 retroelement reported for wallaby (marsupial) interspecific hybrids. The similarity between the two phenomena is consistent with the hypothesis that evolution of the gibbons is characterized by defects in epigenetic repression of TEs, perhaps triggered by interspecific hybridization.
doi:10.1093/gbe/evs048
PMCID: PMC3606032  PMID: 22593550
gibbon; centromere; transposable element; SVA; hybrid
22.  A resource for the conditional ablation of microRNAs in the mouse 
Cell reports  2012;1(4):385-391.
Summary
The importance of miRNAs during development and disease processes is well established. However, most studies have been done in cells or with patient tissues, and therefore the physiological roles of miRNAs are not well understood. To unravel in vivo functions of miRNAs, we have generated conditional, reporter-tagged knockout-first mice for numerous evolutionarily conserved miRNAs. Here we report the generation of 162 miRNA targeting vectors, 64 targeted ES cell lines, and 46 germline-transmitted miRNA knockout mice. In vivo lacZ reporter analysis in 18 lines revealed highly tissue-specific expression patterns and their miRNA expression profiling matched closely with published expression data. Most miRNA knockout mice tested were viable, supporting a mechanism by which miRNAs act redundantly with other miRNAs or other pathways. These data and collection of resources will be of value for the in vivo dissection of miRNA functions in mouse models.
doi:10.1016/j.celrep.2012.02.008
PMCID: PMC3345170  PMID: 22570807
23.  The genome of the green anole lizard and a comparative analysis with birds and mammals 
Nature  2011;477(7366):587-591.
The evolution of the amniotic egg was one of the great evolutionary innovations in the history of life, freeing vertebrates from an obligatory connection to water and thus permitting the conquest of terrestrial environments1. Among amniotes, genome sequences are available for mammals2 and birds3–5, but not for non-avian reptiles. Here we report the genome sequence of the North American green anole lizard, Anolis carolinensis. We find that A. carolinensis microchromosomes are highly syntenic with chicken microchromosomes, yet do not exhibit the high GC and low repeat content that are characteristic of avian microchromosomes3. Also, A. carolinensis mobile elements are very young and diverse – more so than in any other sequenced amniote genome. This lizard genome’s GC content is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds6. We describe and assign sequence to the previously unknown A. carolinensis X chromosome. Comparative gene analysis shows that amniote egg proteins have evolved significantly more rapidly than other proteins. An anole phylogeny resolves basal branches to illuminate the history of their repeated adaptive radiations.
doi:10.1038/nature10390
PMCID: PMC3184186  PMID: 21881562
24.  Deficiencies in lamin B1 and lamin B2 cause neurodevelopmental defects and distinct nuclear shape abnormalities in neurons 
Molecular Biology of the Cell  2011;22(23):4683-4693.
Lamin B1 is essential for neuronal migration and progenitor proliferation during the development of the cerebral cortex. The observation of distinct phenotypes of Lmnb1- and Lmnb2-knockout mice and the differences in the nuclear morphology of cortical neurons in vivo suggest that lamin B1 and lamin B2 play distinct functions in the developing brain.
Neuronal migration is essential for the development of the mammalian brain. Here, we document severe defects in neuronal migration and reduced numbers of neurons in lamin B1–deficient mice. Lamin B1 deficiency resulted in striking abnormalities in the nuclear shape of cortical neurons; many neurons contained a solitary nuclear bleb and exhibited an asymmetric distribution of lamin B2. In contrast, lamin B2 deficiency led to increased numbers of neurons with elongated nuclei. We used conditional alleles for Lmnb1 and Lmnb2 to create forebrain-specific knockout mice. The forebrain-specific Lmnb1- and Lmnb2-knockout models had a small forebrain with disorganized layering of neurons and nuclear shape abnormalities, similar to abnormalities identified in the conventional knockout mice. A more severe phenotype, complete atrophy of the cortex, was observed in forebrain-specific Lmnb1/Lmnb2 double-knockout mice. This study demonstrates that both lamin B1 and lamin B2 are essential for brain development, with lamin B1 being required for the integrity of the nuclear lamina, and lamin B2 being important for resistance to nuclear elongation in neurons.
doi:10.1091/mbc.E11-06-0504
PMCID: PMC3226484  PMID: 21976703
25.  Comparative and demographic analysis of orangutan genomes 
Locke, Devin P. | Hillier, LaDeana W. | Warren, Wesley C. | Worley, Kim C. | Nazareth, Lynne V. | Muzny, Donna M. | Yang, Shiaw-Pyng | Wang, Zhengyuan | Chinwalla, Asif T. | Minx, Pat | Mitreva, Makedonka | Cook, Lisa | Delehaunty, Kim D. | Fronick, Catrina | Schmidt, Heather | Fulton, Lucinda A. | Fulton, Robert S. | Nelson, Joanne O. | Magrini, Vincent | Pohl, Craig | Graves, Tina A. | Markovic, Chris | Cree, Andy | Dinh, Huyen H. | Hume, Jennifer | Kovar, Christie L. | Fowler, Gerald R. | Lunter, Gerton | Meader, Stephen | Heger, Andreas | Ponting, Chris P. | Marques-Bonet, Tomas | Alkan, Can | Chen, Lin | Cheng, Ze | Kidd, Jeffrey M. | Eichler, Evan E. | White, Simon | Searle, Stephen | Vilella, Albert J. | Chen, Yuan | Flicek, Paul | Ma, Jian | Raney, Brian | Suh, Bernard | Burhans, Richard | Herrero, Javier | Haussler, David | Faria, Rui | Fernando, Olga | Darré, Fleur | Farré, Domènec | Gazave, Elodie | Oliva, Meritxell | Navarro, Arcadi | Roberto, Roberta | Capozzi, Oronzo | Archidiacono, Nicoletta | Valle, Giuliano Della | Purgato, Stefania | Rocchi, Mariano | Konkel, Miriam K. | Walker, Jerilyn A. | Ullmer, Brygg | Batzer, Mark A. | Smit, Arian F. A. | Hubley, Robert | Casola, Claudio | Schrider, Daniel R. | Hahn, Matthew W. | Quesada, Victor | Puente, Xose S. | Ordoñez, Gonzalo R. | López-Otín, Carlos | Vinar, Tomas | Brejova, Brona | Ratan, Aakrosh | Harris, Robert S. | Miller, Webb | Kosiol, Carolin | Lawson, Heather A. | Taliwal, Vikas | Martins, André L. | Siepel, Adam | RoyChoudhury, Arindam | Ma, Xin | Degenhardt, Jeremiah | Bustamante, Carlos D. | Gutenkunst, Ryan N. | Mailund, Thomas | Dutheil, Julien Y. | Hobolth, Asger | Schierup, Mikkel H. | Chemnick, Leona | Ryder, Oliver A. | Yoshinaga, Yuko | de Jong, Pieter J. | Weinstock, George M. | Rogers, Jeffrey | Mardis, Elaine R. | Gibbs, Richard A. | Wilson, Richard K.
Nature  2011;469(7331):529-533.
“Orangutan” is derived from the Malay term “man of the forest” and aptly describes the Southeast Asian great apes native to Sumatra and Borneo. The orangutan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orangutan draft genome assembly and short read sequence data from five Sumatran and five Bornean orangutan genomes. Our analyses reveal that, compared to other primates, the orangutan genome has many unique features. Structural evolution of the orangutan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe the first primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orangutan genome structure. Orangutans have extremely low energy usage for a eutherian mammal1, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400k years ago (ya), is more recent than most previous studies and underscores the complexity of the orangutan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (Ne) expanded exponentially relative to the ancestral Ne after the split, while Bornean Ne declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.
doi:10.1038/nature09687
PMCID: PMC3060778  PMID: 21270892

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