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1.
Cell ; 149(4): 912-22, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22559943

RESUMO

Gene duplication is an important source of phenotypic change and adaptive evolution. We leverage a haploid hydatidiform mole to identify highly identical sequences missing from the reference genome, confirming that the cortical development gene Slit-Robo Rho GTPase-activating protein 2 (SRGAP2) duplicated three times exclusively 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 analyses show that SRGAP2C is the most likely duplicate to encode a functional protein and is among the most fixed human-specific duplicate genes. Our data suggest a mechanism where incomplete duplication created a novel gene function-antagonizing parental SRGAP2 function-immediately "at birth" 2-3 mya, which is a time corresponding to the transition from Australopithecus to Homo and the beginning of neocortex expansion.


Assuntos
Evolução Molecular , Proteínas Ativadoras de GTPase/genética , Primatas/genética , Duplicações Segmentares Genômicas , Animais , Variações do Número de Cópias de DNA , Feminino , Genética Médica , Humanos , Mola Hidatiforme/genética , Hibridização in Situ Fluorescente , Mamíferos/genética , Dados de Sequência Molecular , Gravidez
2.
J Lipid Res ; 61(3): 413-421, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31941672

RESUMO

Zinc metallopeptidase STE24 (ZMPSTE24) is essential for the conversion of farnesyl-prelamin A to mature lamin A, a key component of the nuclear lamina. In the absence of ZMPSTE24, farnesyl-prelamin A accumulates in the nucleus and exerts toxicity, causing a variety of disease phenotypes. By ∼4 months of age, both male and female Zmpste24-/- mice manifest a near-complete loss of adipose tissue, but it has never been clear whether this phenotype is a direct consequence of farnesyl-prelamin A toxicity in adipocytes. To address this question, we generated a conditional knockout Zmpste24 allele and used it to create adipocyte-specific Zmpste24-knockout mice. To boost farnesyl-prelamin A levels, we bred in the "prelamin A-only" Lmna allele. Gene expression, immunoblotting, and immunohistochemistry experiments revealed that adipose tissue in these mice had decreased Zmpste24 expression along with strikingly increased accumulation of prelamin A. In male mice, Zmpste24 deficiency in adipocytes was accompanied by modest changes in adipose stores (an 11% decrease in body weight, a 23% decrease in body fat mass, and significantly smaller gonadal and inguinal white adipose depots). No changes in adipose stores were detected in female mice, likely because prelamin A expression in adipose tissue is lower in female mice. Zmpste24 deficiency in adipocytes did not alter the number of macrophages in adipose tissue, nor did it alter plasma levels of glucose, triglycerides, or fatty acids. We conclude that ZMPSTE24 deficiency in adipocytes, and the accompanying accumulation of farnesyl-prelamin A, reduces adipose tissue stores, but only modestly and only in male mice.


Assuntos
Tecido Adiposo/metabolismo , Lamina Tipo A/metabolismo , Proteínas de Membrana/metabolismo , Metaloendopeptidases/metabolismo , Tecido Adiposo/química , Alelos , Animais , Núcleo Celular/química , Núcleo Celular/metabolismo , Feminino , Masculino , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Metaloendopeptidases/deficiência , Metaloendopeptidases/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos
3.
Nature ; 513(7517): 195-201, 2014 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-25209798

RESUMO

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.


Assuntos
Genoma/genética , Hylobates/classificação , Hylobates/genética , Cariótipo , Filogenia , Animais , Evolução Molecular , Hominidae/classificação , Hominidae/genética , Humanos , Dados de Sequência Molecular , Retroelementos/genética , Seleção Genética , Terminação da Transcrição Genética
4.
PLoS Genet ; 12(2): e1005691, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26839965

RESUMO

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.


Assuntos
Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Marcação de Genes , Mutação/genética , Animais , Regulação para Baixo/genética , Deleção de Genes , Biblioteca Gênica , Genoma , Homozigoto , Camundongos Endogâmicos C57BL , Regulação para Cima/genética
5.
Genome Res ; 25(4): 598-607, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25591789

RESUMO

Expression of the bacterial beta-galactosidase reporter gene (lacZ) in the vector used for the Knockout Mouse Project (KOMP) is driven by the endogenous promoter of the target gene. In tissues from KOMP mice, histochemical staining for LacZ enzyme activity can be used to determine gene expression patterns. With this technique, we have produced a comprehensive resource of gene expression using both whole mount (WM) and frozen section (FS) LacZ staining in 313 unique KOMP mutant mouse lines. Of these, ∼ 80% of mutants showed specific staining in one or more tissues, while ∼ 20% showed no specific staining, ∼ 13% had staining in only one tissue, and ∼ 25% had staining in >6 tissues. The highest frequency of specific staining occurred in the brain (∼ 50%), male gonads (42%), and kidney (39%). The WM method was useful for rapidly identifying whole organ and some substructure staining, while the FS method often revealed substructure and cellular staining specificity. Both staining methods had >90% repeatability in biological replicates. Nonspecific LacZ staining occurs in some tissues due to the presence of bacteria or endogenous enzyme activity. However, this can be effectively distinguished from reporter gene activity by the combination of the WM and FS methods. After careful annotation, LacZ staining patterns in a high percentage of mutants revealed a unique structure-function not previously reported for many of these genes. The validation of methods for LacZ staining, annotation, and expression analysis reported here provides unique insights into the function of genes for which little is currently known.


Assuntos
Regulação da Expressão Gênica/genética , Genes Reporter/genética , Óperon Lac/genética , Regiões Promotoras Genéticas/genética , Animais , Atlas como Assunto , Feminino , Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coloração e Rotulagem , Relação Estrutura-Atividade
6.
J Lipid Res ; 58(7): 1453-1461, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28476858

RESUMO

Mutation of conserved cysteines in proteins of the Ly6 family cause human disease-chylomicronemia in the case of glycosylphosphatidylinositol-anchored HDL binding protein 1 (GPIHBP1) and paroxysmal nocturnal hemoglobinuria in the case of CD59. A mutation in a conserved cysteine in CD59 prevented the protein from reaching the surface of blood cells. In contrast, mutation of conserved cysteines in human GPIHBP1 had little effect on GPIHBP1 trafficking to the surface of cultured CHO cells. The latter findings were somewhat surprising and raised questions about whether CHO cell studies accurately model the fate of mutant GPIHBP1 proteins in vivo. To explore this concern, we created mice harboring a GPIHBP1 cysteine mutation (p.C63Y). The p.C63Y mutation abolished the ability of mouse GPIHBP1 to bind LPL, resulting in severe chylomicronemia. The mutant GPIHBP1 was detectable by immunohistochemistry on the surface of endothelial cells, but the level of expression was ∼70% lower than in WT mice. The mutant GPIHBP1 protein in mouse tissues was predominantly monomeric. We conclude that mutation of a conserved cysteine in GPIHBP1 abolishes the ability of GPIHBP1 to bind LPL, resulting in mislocalization of LPL and severe chylomicronemia. The mutation reduced but did not eliminate GPIHBP1 on the surface of endothelial cells in vivo.


Assuntos
Sequência Conservada , Cisteína , Lipase Lipoproteica/metabolismo , Mutação , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Feminino , Humanos , Lipase Lipoproteica/genética , Camundongos , Ligação Proteica/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Lipoproteínas/genética , Triglicerídeos/sangue
7.
Transgenic Res ; 26(2): 263-277, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27905063

RESUMO

The CRISPR/Cas9 system has rapidly advanced targeted genome editing technologies. However, its efficiency in targeting with constructs in mouse zygotes via homology directed repair (HDR) remains low. Here, we systematically explored optimal parameters for targeting constructs in mouse zygotes via HDR using mouse embryonic stem cells as a model system. We characterized several parameters, including single guide RNA cleavage activity and the length and symmetry of homology arms in the construct, and we compared the targeting efficiency between Cas9, Cas9nickase, and dCas9-FokI. We then applied the optimized conditions to zygotes, delivering Cas9 as either mRNA or protein. We found that Cas9 nucleo-protein complex promotes highly efficient, multiplexed targeting of circular constructs containing reporter genes and floxed exons. This approach allows for a one-step zygote injection procedure targeting multiple genes to generate conditional alleles via homologous recombination, and simultaneous knockout of corresponding genes in non-targeted alleles via non-homologous end joining.


Assuntos
Sistemas CRISPR-Cas/genética , Marcação de Genes/métodos , Recombinação Homóloga/genética , Alelos , Animais , Reparo do DNA por Junção de Extremidades/genética , Éxons , Camundongos , RNA Guia de Cinetoplastídeos/genética , RNA Mensageiro/genética , Zigoto/crescimento & desenvolvimento
8.
Nature ; 474(7351): 337-42, 2011 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-21677750

RESUMO

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.


Assuntos
Deleção de Genes , Técnicas de Inativação de Genes/métodos , Genes/genética , Estudos de Associação Genética/métodos , Genoma/genética , Camundongos Knockout/genética , Alelos , Animais , Biologia Computacional , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genes Letais/genética , Vetores Genéticos/genética , Genômica , Genótipo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese Insercional/métodos , Fenótipo , Reação em Cadeia da Polimerase , Ratos
9.
Nature ; 477(7366): 587-91, 2011 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-21881562

RESUMO

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 environments. Among amniotes, genome sequences are available for mammals and birds, 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 microchromosomes. Also, A. carolinensis mobile elements are very young and diverse-more so than in any other sequenced amniote genome. The GC content of this lizard genome is also unusual in its homogeneity, unlike the regionally variable GC content found in mammals and birds. 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.


Assuntos
Aves/genética , Evolução Molecular , Genoma/genética , Lagartos/genética , Mamíferos/genética , Animais , Galinhas/genética , Sequência Rica em GC/genética , Genômica , Humanos , Dados de Sequência Molecular , Filogenia , Sintenia/genética , Cromossomo X/genética
10.
Proc Natl Acad Sci U S A ; 110(21): E1923-32, 2013 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-23650370

RESUMO

The role of protein farnesylation in lamin A biogenesis and the pathogenesis of progeria has been studied in considerable detail, but the importance of farnesylation for the B-type lamins, lamin B1 and lamin B2, has received little attention. Lamins B1 and B2 are expressed in nearly every cell type from the earliest stages of development, and they have been implicated in a variety of functions within the cell nucleus. To assess the importance of protein farnesylation for B-type lamins, we created knock-in mice expressing nonfarnesylated versions of lamin B1 and lamin B2. Mice expressing nonfarnesylated lamin B2 developed normally and were free of disease. In contrast, mice expressing nonfarnesylated lamin B1 died soon after birth, with severe neurodevelopmental defects and striking nuclear abnormalities in neurons. The nuclear lamina in migrating neurons was pulled away from the chromatin so that the chromatin was left "naked" (free from the nuclear lamina). Thus, farnesylation of lamin B1--but not lamin B2--is crucial for brain development and for retaining chromatin within the bounds of the nuclear lamina during neuronal migration.


Assuntos
Encéfalo/embriologia , Movimento Celular/fisiologia , Cromatina/metabolismo , Lamina Tipo B/metabolismo , Lâmina Nuclear/metabolismo , Prenilação de Proteína/fisiologia , Animais , Cromatina/genética , Lamina Tipo B/genética , Camundongos , Camundongos Transgênicos , Lâmina Nuclear/genética
11.
Genome Res ; 22(12): 2520-8, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22892276

RESUMO

Chromosome rearrangements in small apes are up to 20 times more frequent than in most mammals. Because of their complexity, the full extent of chromosome evolution in these hominoids is not yet fully documented. However, previous work with array painting, BAC-FISH, and selective sequencing in two of the four karyomorphs has shown that high-resolution methods can precisely define chromosome breakpoints and map the complex flow of evolutionary chromosome rearrangements. Here we use these tools to precisely define the rearrangements that have occurred in the remaining two karyomorphs, genera Symphalangus (2n = 50) and Hoolock (2n = 38). This research provides the most comprehensive insight into the evolutionary origins of chromosome rearrangements involved in transforming small apes genome. Bioinformatics analyses of the human-gibbon synteny breakpoints revealed association with transposable elements and segmental duplications, providing some insight into the mechanisms that might have promoted rearrangements in small apes. In the near future, the comparison of gibbon genome sequences will provide novel insights to test hypotheses concerning the mechanisms of chromosome evolution. The precise definition of synteny block boundaries and orientation, chromosomal fusions, and centromere repositioning events presented here will facilitate genome sequence assembly for these close relatives of humans.


Assuntos
Aberrações Cromossômicas , Cromossomos/genética , Análise Citogenética/métodos , Rearranjo Gênico , Hylobates/genética , Animais , Centrômero/química , Centrômero/genética , Elementos de DNA Transponíveis , Bases de Dados Genéticas , Evolução Molecular , Feminino , Humanos , Hibridização in Situ Fluorescente , Cariótipo , Mutação , Filogenia
12.
Proc Natl Acad Sci U S A ; 109(37): E2486-95, 2012 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-22908270

RESUMO

The three lipin phosphatidate phosphatase (PAP) enzymes catalyze a step in glycerolipid biosynthesis, the conversion of phosphatidate to diacylglycerol. Lipin-1 is critical for lipid synthesis and homeostasis in adipose tissue, liver, muscle, and peripheral nerves. Little is known about the physiological role of lipin-2, the predominant lipin protein present in liver and the deficient gene product in the rare disorder Majeed syndrome. By using lipin-2-deficient mice, we uncovered a functional relationship between lipin-1 and lipin-2 that operates in a tissue-specific and age-dependent manner. In liver, lipin-2 deficiency led to a compensatory increase in hepatic lipin-1 protein and elevated PAP activity, which maintained lipid homeostasis under basal conditions, but led to diet-induced hepatic triglyceride accumulation. As lipin-2-deficient mice aged, they developed ataxia and impaired balance. This was associated with the combination of lipin-2 deficiency and an age-dependent reduction in cerebellar lipin-1 levels, resulting in altered cerebellar phospholipid composition. Similar to patients with Majeed syndrome, lipin-2-deficient mice developed anemia, but did not show evidence of osteomyelitis, suggesting that additional environmental or genetic components contribute to the bone abnormalities observed in patients. Combined lipin-1 and lipin-2 deficiency caused embryonic lethality. Our results reveal functional interactions between members of the lipin family in vivo, and a unique role for lipin-2 in central nervous system biology that may be particularly important with advancing age. Additionally, as has been observed in mice and humans with lipin-1 deficiency, the pathophysiology in lipin-2 deficiency is associated with dysregulation of lipid intermediates.


Assuntos
Envelhecimento/fisiologia , Cerebelo/fisiologia , Homeostase/fisiologia , Fígado/fisiologia , Proteínas Nucleares/metabolismo , Fosfatidato Fosfatase/metabolismo , Análise de Variância , Animais , Contagem de Células Sanguíneas , Western Blotting , Osso e Ossos/diagnóstico por imagem , Cerebelo/metabolismo , Primers do DNA/genética , Galactosídeos , Perfilação da Expressão Gênica , Técnicas Histológicas , Imuno-Histoquímica , Indóis , Fígado/metabolismo , Locomoção/fisiologia , Camundongos , Camundongos Transgênicos , Proteínas Nucleares/deficiência , Fosfatidato Fosfatase/deficiência , Fosfolipídeos/metabolismo , Reação em Cadeia da Polimerase , Desempenho Psicomotor , Radiografia , Reflexo de Sobressalto/fisiologia
13.
Genome Res ; 21(3): 402-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21282478

RESUMO

In Mus spretus, the chloride channel 4 gene Clcn4-2 is X-linked and dosage compensated by X up-regulation and X inactivation, while in the closely related mouse species Mus musculus, Clcn4-2 has been translocated to chromosome 7. We sequenced Clcn4-2 in M. spretus and identified the breakpoints of the evolutionary translocation in the Mus lineage. Genetic and epigenetic differences were observed between the 5'ends of the autosomal and X-linked loci. Remarkably, Clcn4-2 introns have been truncated on chromosome 7 in M. musculus as compared with the X-linked loci from seven other eutherian mammals. Intron sequences specifically preserved in the X-linked loci were significantly enriched in AT-rich oligomers. Genome-wide analyses showed an overall enrichment in AT motifs unique to the eutherian X (except for genes that escape X inactivation), suggesting a role for these motifs in regulation of the X chromosome.


Assuntos
Canais de Cloreto/genética , Região 5'-Flanqueadora/genética , Sequência Rica em At , Animais , Sequência de Bases , Canais de Cloreto/metabolismo , Quebra Cromossômica , Mapeamento Cromossômico , Mecanismo Genético de Compensação de Dose , Epigenômica , Evolução Molecular , Feminino , Dosagem de Genes , Genes , Genoma , Humanos , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , Muridae , Estrutura Terciária de Proteína/genética , Ratos , Análise de Sequência de DNA , Especificidade da Espécie , Cromossomo X/genética
14.
Nature ; 453(7198): 1064-71, 2008 Jun 19.
Artigo em Inglês | MEDLINE | ID: mdl-18563158

RESUMO

Lancelets ('amphioxus') are the modern survivors of an ancient chordate lineage, with a fossil record dating back to the Cambrian period. Here we describe the structure and gene content of the highly polymorphic approximately 520-megabase genome of the Florida lancelet Branchiostoma floridae, and analyse it in the context of chordate evolution. Whole-genome comparisons illuminate the murky relationships among the three chordate groups (tunicates, lancelets and vertebrates), and allow not only reconstruction of the gene complement of the last common chordate ancestor but also partial reconstruction of its genomic organization, as well as a description of two genome-wide duplications and subsequent reorganizations in the vertebrate lineage. These genome-scale events shaped the vertebrate genome and provided additional genetic variation for exploitation during vertebrate evolution.


Assuntos
Cordados/genética , Evolução Molecular , Genoma/genética , Animais , Cordados/classificação , Sequência Conservada , Elementos de DNA Transponíveis/genética , Duplicação Gênica , Genes/genética , Ligação Genética , Humanos , Íntrons/genética , Cariotipagem , Família Multigênica , Filogenia , Polimorfismo Genético/genética , Proteínas/genética , Sintenia , Fatores de Tempo , Vertebrados/classificação , Vertebrados/genética
15.
Mol Biol Evol ; 29(11): 3441-50, 2012 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22683814

RESUMO

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.


Assuntos
Elementos Alu/genética , Hylobates/genética , Filogenia , Animais , Sudeste Asiático , Biologia Computacional , Mineração de Dados , Loci Gênicos/genética , Genoma/genética , Geografia , Humanos , Dados de Sequência Molecular , Mutagênese Insercional/genética , Reação em Cadeia da Polimerase
16.
Hum Mol Genet ; 20(18): 3537-44, 2011 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-21659336

RESUMO

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 (Lmnb1(Δ/Δ)Lmnb2(Δ/Δ)). Lmnb1 and Lmnb2 transcripts were absent in keratinocytes of Lmnb1(Δ/Δ)Lmnb2(Δ/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 Lmnb1(Δ/Δ)Lmnb2(Δ/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 Lmnb1(Δ/Δ)Lmnb2(Δ/Δ) mice. Lmnb1(Δ/Δ)Lmnb2(Δ/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.


Assuntos
Proliferação de Células , Cabelo/crescimento & desenvolvimento , Queratinócitos/citologia , Lamina Tipo B/deficiência , Pele/crescimento & desenvolvimento , Células 3T3 , Animais , Células Cultivadas , Feminino , Cabelo/metabolismo , Células HeLa , Humanos , Queratinócitos/metabolismo , Lamina Tipo B/genética , Masculino , Camundongos , Camundongos Knockout , Pele/metabolismo
17.
Nat Genet ; 36(9): 955-7, 2004 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-15300250

RESUMO

CHARGE syndrome is a common cause of congenital anomalies affecting several tissues in a nonrandom fashion. We report a 2.3-Mb de novo overlapping microdeletion on chromosome 8q12 identified by array comparative genomic hybridization in two individuals with CHARGE syndrome. Sequence analysis of genes located in this region detected mutations in the gene CHD7 in 10 of 17 individuals with CHARGE syndrome without microdeletions, accounting for the disease in most affected individuals.


Assuntos
Anormalidades Múltiplas/genética , Atresia das Cóanas/genética , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Cardiopatias Congênitas/genética , Mutação , Coloboma/genética , Surdez/genética , Deleção de Genes , Humanos , Análise de Sequência de DNA , Síndrome
18.
Mol Biol Evol ; 28(8): 2211-8, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21368318

RESUMO

Gibbons are small, arboreal, highly endangered apes that are understudied compared with other hominoids. At present, there are four recognized genera and approximately 17 species, all likely to have diverged from each other within the last 5-6 My. Although the gibbon phylogeny has been investigated using various approaches (i.e., vocalization, morphology, mitochondrial DNA, karyotype, etc.), the precise taxonomic relationships are still highly debated. Here, we present the first survey of nuclear sequence variation within and between gibbon species with the goal of estimating basic population genetic parameters. We gathered ~60 kb of sequence data from a panel of 19 gibbons representing nine species and all four genera. We observe high levels of nucleotide diversity within species, indicative of large historical population sizes. In addition, we find low levels of genetic differentiation between species within a genus comparable to what has been estimated for human populations. This is likely due to ongoing or episodic gene flow between species, and we estimate a migration rate between Nomascus leucogenys and N. gabriellae of roughly one migrant every two generations. Together, our findings suggest that gibbons have had a complex demographic history involving hybridization or mixing between diverged populations.


Assuntos
Variação Genética/genética , Hylobates/genética , Animais , Linhagem Celular , Fluxo Gênico/genética , Ligação Genética , Hylobates/classificação , Mutação/genética , Fenótipo , Filogenia
19.
Mamm Genome ; 23(9-10): 580-6, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22968824

RESUMO

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.


Assuntos
Camundongos Knockout/genética , Animais , Internacionalidade , Internet , Camundongos
20.
Nat Methods ; 6(6): 431-4, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19465919

RESUMO

We constructed Drosophila melanogaster bacterial artificial chromosome libraries with 21-kilobase and 83-kilobase inserts in the P[acman] system. We mapped clones representing 12-fold coverage and encompassing more than 95% of annotated genes onto the reference genome. These clones can be integrated into predetermined attP sites in the genome using UC31 integrase to rescue mutations. They can be modified through recombineering, for example, to incorporate protein tags and assess expression patterns.


Assuntos
Animais Geneticamente Modificados/genética , Mapeamento Cromossômico/métodos , Cromossomos Artificiais Bacterianos/genética , Clonagem Molecular/métodos , Drosophila melanogaster/genética , Biblioteca Gênica , Animais , Sequência de Bases , Dados de Sequência Molecular
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