RESUMO
The purpose of this study was to identify and classify endogenous retroviruses (ERVs) in the cat genome. Pooled DNA from five domestic cats was subjected to degenerate PCR with primers specific to the conserved retroviral pro/pol region. The 59 amplified retroviral sequences were used for in silico analysis of the cat genome (Felis_catus-6.2). We identified 219 ERV γ and ß elements from cat genome contigs, which were classified into 42 ERV γ and 4 ß families and further analysed. Among them, 99 γ and 5 ß ERV elements contained the complete retroviral structure. Furthermore, we identified 757 spuma-like ERV elements based on the sequence homology to murine (Mu)ERV-L and human (H)ERV-L. To the best of our knowledge, this is the first detailed genome-scale analysis examining Felis catus endogenous retroviruses (FcERV) and providing advanced insights into their structural characteristics, localization in the genome, and diversity.
Assuntos
Gatos/virologia , Simulação por Computador , Retrovirus Endógenos/classificação , Retrovirus Endógenos/genética , Genoma , Reação em Cadeia da Polimerase/métodos , Animais , Gatos/genética , Primers do DNA , Produtos do Gene pol/química , Produtos do Gene pol/genética , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNARESUMO
Primary cilia exert a profound impact on cell signalling and cell cycle progression. Recently, actin cytoskeleton destabilization has been recognized as a dominant inducer of ciliogenesis, but the exact mechanisms regulating ciliogenesis remain poorly understood. Here we show that the actin cytoskeleton remodelling controls ciliogenesis by regulating transcriptional coactivator YAP/TAZ as well as ciliary vesicle trafficking. Cytoplasmic retention of YAP/TAZ correlates with active ciliogenesis either in spatially confined cells or in cells treated with an actin filament destabilizer. Moreover, knockdown of YAP/TAZ is sufficient to induce ciliogenesis, whereas YAP/TAZ hyperactivation suppresses serum starvation-mediated ciliogenesis. We also identify actin remodelling factors LIMK2 and TESK1 as key players in the ciliogenesis control network in which YAP/TAZ and directional vesicle trafficking are integral components. Our work provides new insights for understanding the link between actin dynamics and ciliogenesis.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cílios/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fosfoproteínas/metabolismo , Western Blotting , Linhagem Celular , Imunofluorescência , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Quinases Lim/metabolismo , Microscopia de Fluorescência , Proteínas Serina-Treonina Quinases/metabolismo , Transativadores , Fatores de Transcrição , Ativação Transcricional , Proteínas com Motivo de Ligação a PDZ com Coativador Transcricional , Proteínas de Sinalização YAPRESUMO
Pooled genomic DNA from 10 dogs was subjected to polymerase chain reaction with primers targeting the retroviral pro/pol region. Sequence analysis of 120 clones obtained by PCR revealed 81 of retroviral origin. Subsequent analysis of the dog genome (CanFam 2.0) by BLAST investigation using degenerate PCR products and previously identified retroviral sequences permitted the identification of additional retroviral γ and ß sequences. A phylogenetic analysis using the retroviral protease (PR) and reverse transcriptase (RT) sequences in the dog genome resulted in identification of 17 γ and 7 ß families. In addition, we also identified 167 spuma-like ERV elements from CanFam 2.0 based on sequence homology to murine (Mu)ERV-L and human (H)ERV-L. Our results could contribute to the understanding of the influence of retroviruses in shaping the genome structure and altering gene expression by providing quantitative and locational information of ERV loci and their diversity in the dog genome.
Assuntos
Betaretrovirus/genética , Cães/genética , Cães/virologia , Retrovirus Endógenos/genética , Gammaretrovirus/genética , Reação em Cadeia da Polimerase/veterinária , Sequência de Aminoácidos , Animais , Betaretrovirus/classificação , Retrovirus Endógenos/classificação , Gammaretrovirus/classificação , Genoma , Dados de Sequência Molecular , Filogenia , Reação em Cadeia da Polimerase/métodos , Software , Spumavirus/classificação , Spumavirus/genética , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Homozygous Purkinje cell degeneration (pcd) mutant males exhibit abnormal sperm development. Microscopic examination of the testes from pcd(3J)-/- mice at postnatal days 12, 15, 18 and 60 revealed histological differences, in comparison to wild-type mice, which were evident by day 18. Greatly reduced numbers of spermatocytes and spermatids were found in the adult testes, and apoptotic cells were identified among the differentiating germ cells after day 15. Our immunohistological analysis using an antihuman AGTPBP1 antibody showed that AGTPBP1 was expressed in spermatogenic cells between late stage primary spermatocytes and round spermatids. A global gene expression analysis from the testes of pcd(3J)-/- mice showed that expression of cyclin B3 and de-ubiquitinating enzymes USP2 and USP9y was altered by >1.5-fold compared to the expression levels in the wild-type. Our results suggest that the pcd mutant mice have defects in spermatogenesis that begin with the pachytene spermatocyte stage and continue through subsequent stages. Thus, Agtpbp1, the gene responsible for the pcd phenotype, plays an important role in spermatogenesis and is important for survival of germ cells at spermatocytes stage onward.