Analysis of Chromatin-associated Fiber Arrays

Electron microscopic examination of chromatin from embryonic nuclei of Oncopeltus fasciatus and Drosophila melanogaster reveals arrays of chromatin associated fibers. The lengths and spacings of these fibers were analyzed to provide a basis for defining and interpreting regions of transcriptionally active chromatin. The results of the analysis are consistent with the interpretation of some fibers as nascent RNA with associated protein (RNP). The chromatin segments underlying these fiber arrays were classified as ribosomal or non-ribosomal transcription units according to definitions and criteria described by Foe et al. (1976). Nascent fibers on active ribosomal transcription units were analyzed and compared for Drosophila melanogaster, Triturus viridescens, and Oncopeltus fasciatus. A common feature of the fiber patterns on ribosomal TUs is that origin-distal fibers exhibit greater length variability and a lower slope relative to proximal fibers. The region of increased variability in fiber lengths is correlated with the expected location of 28S ribosomal RNA sequences in the distal half of each ribosomal transcription unit. Because 28S ribosomal RNA appears to contain more extensive regions of base sequence complementarity, we suggest that the length of ribosomal RNP fibers is influenced under our spreading conditions by the secondary structure of the nascent RNA. In order to calculate the RNA content of RNP fibers, chromatin morphology was used to estimate lengths of transcribed DNA. The packing ratio of DNA in chromatin, which we express as the length of B-structure DNA divided by length of chromatin, is 1.1-1.2 and 1.6 for the DNA in active ribosomal and non-ribosomal chromatins, respectively. These DNA packing ratios are used to determine the extent to which nascent RNP fibers are shorter than the transcribed DNA (expressed as DNA/RNP length ratio). For non-ribosomal transcription units and for proximal fibers of ribosomal transcription units. DNA/RNP length ratios are relatively constant within each array. However, considerable variability in this ratio (4-23) is observed for different arrays of fibers. Possible sources of this variability are considered by comparing ratios derived from the presumably identical ribosomal transcription units. Further analysis of the morphology of nascent fibers may elucidate the contributions of proteins and successive RNA sequences to RNP structure.