RESUMEN
This paper describes image processing methods for automatic spotted microarray image analysis. Automatic gridding is important to achieve constant data quality and is, therefore, especially interesting for large-scale experiments as well as for integration of microarray expression data from different sources. We propose a Markov random field (MRF) based approach to high-level grid segmentation, which is robust to common problems encountered with array images and does not require calibration. We also propose an active contour method for single-spot segmentation. Active contour models describe objects in images by properties of their boundaries. Both MRFs and active contour models have been used in various other computer vision applications. The traditional active contour model must be generalized for successful application to microarray spot segmentation. Our active contour model is employed for spot detection in the MRF score functions as well as for spot signal segmentation in quantitative array image analysis. An evaluation using several image series from different sources shows the robustness of our methods.
Asunto(s)
Algoritmos , ADN/análisis , Aumento de la Imagen/métodos , Interpretación de Imagen Asistida por Computador/métodos , Microscopía Fluorescente/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Robótica/métodos , ADN/química , ADN/genética , Perfilación de la Expresión Génica/métodos , Cadenas de Markov , Modelos Genéticos , Modelos Estadísticos , Nanotecnología/métodos , Reconocimiento de Normas Patrones Automatizadas , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Análisis de Secuencia de ADN/métodosRESUMEN
Germ plasm formation is considered to define the first step in germ cell development. Xenopus Dead end represents a germ plasm specific transcript that is homologous to the previously characterized zebrafish dead end, which is required for germ cell migration and survival. XDead end mRNA localizes to the vegetal pole of Xenopus oocytes; in contrast to all other known germ plasm associated transcripts in Xenopus, XDead end is transported via the late transport pathway, suggesting a different mode of germ plasm restriction. Vegetal localization in the oocyte is achieved via a localization element mapping to a 251 nucleotide element in the 3'-UTR. This RNA sequence binds to a set of proteins characteristic for the late localization pathway and to one additional protein of 38 kDa. Inhibition of XDead end translation in Xenopus embryos results in a loss of primordial germ cells at tadpole stages of development. Early specification events do not seem to be affected, but the primordial germ cells fail to migrate dorsally and eventually disappear. This phenotype is very similar to what has been observed in the zebrafish, indicating that the role of XDead end in germ cell development has been conserved in evolution.
Asunto(s)
Células Germinativas/fisiología , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Regiones no Traducidas 3'/genética , Regiones no Traducidas 3'/metabolismo , Animales , Movimiento Celular , Embrión no Mamífero/citología , Embrión no Mamífero/fisiología , Femenino , Biblioteca de Genes , Células Germinativas/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Oocitos/metabolismo , Oocitos/fisiología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo , Proteínas de Pez Cebra/genéticaRESUMEN
Receptor-mediated nucleocytoplasmic transport of clock proteins is an important, conserved element of the core mechanism for circadian rhythmicity. A systematic analysis of the nuclear export characteristics for the different murine period (mPER) and cryptochrome (mCRY) proteins using Xenopus oocytes as an experimental system demonstrates that all three mPER proteins, but neither mCRY1 nor mCRY2, are exported if injected individually. However, nuclear injection of heterodimeric complexes that contain combinations of mPER and mCRY proteins shows that mPER1 serves as an export adaptor for mCRY1 and mCRY2. Functional analysis of dominant-negative mPER1 variants designed either to sequester mPER3 to the cytoplasm or to inhibit nuclear export of mCRY1/2 in synchronized, stably transfected fibroblasts suggests that mPER1-mediated export of mCRY1/2 defines an important new element of the core clock machinery in vertebrates.