The organization of animal cell nuclei

Carnegie Institution Of Washington, D.C., Washington DC

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Abstract

Project Summary The long-term goal of our research is to understand the functional organization of the animal cell nucleus. Specifically, we will concentrate on transcription (RNA synthesis) and splicing in the giant ?lampbrush? chromosomes found in oocytes of frogs and salamanders. Two features make these chromosomes ideal for our studies. First, they are the largest known chromosomes, with individual lengths up to 1 mm. Second, they are involved in active transcription and splicing. The transcribing regions appear as hundreds of chromatin loops, each of which contain one or more active genes. The loops themselves are so large (10s to 100s of mm in length) that one can visualize transcription and splicing of specific genes by conventional light microscopy. Our aim now is to analyze the detailed molecular organization of specific transcription units in lampbrush chromosome loops by a combination of 1) single molecule fluorescent in situ hybridization (FISH), 2) deep sequencing of nascent transcripts on the lampbrush chromosomes, 3) deep sequencing of lampbrush chromatin (ChIP-seq), and 4) super-resolution microscopy of specific loops. The aim is to analyze transcription and splicing on a sufficient number of specific chromosome loops that we can draw inferences about the overall organization of active chromatin. These studies could reveal aspects of RNA transcription and processing that are missed by conventional in vitro molecular studies.