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1.
J Genet ; 97(5): 1315-1325, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30555080

RESUMO

Nodal-related protein (ndr2) is amember of the transforming growth factor type ß superfamily of factors and is required for ventral midline patterning of the embryonic central nervous system in zebrafish. In humans, mutations in the gene encoding nodal cause holoprosencephaly and heterotaxy. Mutations in the ndr2 gene in the zebrafish (Danio rerio) lead to similar phenotypes, including loss of the medial floor plate, severe deficits in ventral forebrain development and cyclopia. Alleles of the ndr2 gene have been useful in studying patterning of ventral structures of the central nervous system. Fifteen different ndr2 alleles have been reported in zebrafish, of which eight were generated using chemical mutagenesis, four were radiation-induced and the remaining alleles were obtained via random insertion, gene targeting (TALEN) or unknown methods. Therefore, most mutation sites were random and could not be predicted a priori. Using the CRISPR-Cas9 system from Streptococcus pyogenes, we targeted distinct regions in all three exons of zebrafish ndr2 and observed cyclopia in the injected (G0) embryos.We show that the use of sgRNA-Cas9 ribonucleoprotein (RNP) complexes can cause penetrant cyclopic phenotypes in injected (G0) embryos. Targeted polymerase chain reaction amplicon analysis using Sanger sequencing showed that most of the alleles had small indels resulting in frameshifts. The sequence information correlates with the loss of ndr2 activity. In this study, we validate multiple CRISPR targets using an in vitro nuclease assay and in vivo analysis using embryos. We describe one specific mutant allele resulting in the loss of conserved terminal cysteine-coding sequences. This study is another demonstration of the utility of the CRISPR-Cas9 system in generating domain-specific mutations and provides further insights into the structure-function of the ndr2 gene.


Assuntos
Sistemas CRISPR-Cas , Peptídeos e Proteínas de Sinalização Intracelular/genética , Mutação , Ribonucleoproteínas/genética , Proteínas de Peixe-Zebra/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Holoprosencefalia/genética , Peptídeos e Proteínas de Sinalização Intracelular/química , Modelos Moleculares , Fenótipo , Domínios Proteicos , Ribonucleoproteínas/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química
2.
Curr Biol ; 21(1): 53-8, 2011 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-21167713

RESUMO

Increasing evidence suggests that histone posttranslational modifications (PTMs) function in a combinatorial fashion to regulate the diverse activities associated with chromatin. Yet how these patterns of histone PTMs influence the adapter proteins known to bind them is poorly understood. In addition, how histone-specific antibodies are influenced by these same patterns of PTMs is largely unknown. Here we examine the binding properties of histone-specific antibodies and histone-interacting proteins using peptide arrays containing a library of combinatorially modified histone peptides. We find that modification-specific antibodies are more promiscuous in their PTM recognition than expected and are highly influenced by neighboring PTMs. Furthermore, we find that the binding of histone-interaction domains from BPTF, CHD1, and RAG2 to H3 lysine 4 trimethylation is also influenced by combinatorial PTMs. These results provide further support for the histone code hypothesis and raise specific concerns with the quality of the currently available modification-specific histone antibodies.


Assuntos
Anticorpos/metabolismo , Histonas/imunologia , Histonas/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sequência de Aminoácidos , Afinidade de Anticorpos , Metilação , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína
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