Search

In the nurse cells of Drosophila, nuclear DNA is replicated many times without nuclear division. Nurse cells differ from salivary gland cells, another type of endoreplicated Drosophila cell, in that banded polytene chromosomes are not seen in large nurse cells. Cytophotometry of Feulgen stained nurse cell nuclei that have also been labeled with 3H-thymidine shows that the DNA contents between S-phases are not doublings of the diploid value. In situ hybridization of cloned probes for 28S + 18S ribosomal RNA, 5S RNA, and histone genes, and for satellite, copia, and telomere sequences shows that satellite and histone sequences replicate only partially during nurse cell growth, while 5S sequences fully replicate. However, during the last nurse cell endoreplication cycle, all sequences including the previously under-replicated satellite sequences replicate fully. In situ hybridization experiments also demonstrate that the loci for the multiple copies of histone and 5S RNA genes are clustered into a small number of sites. In contrast, 28S + 18S rRNA genes are dispersed. We discuss the implications of the observed distribution of sequences within nurse cell nuclei for interphase nuclear organization. In the ovarian follicle cells, which undergo only two or three endoreplication cycles, satellite, histone and ribosomal DNA sequences are also found by in situ hybridization to be underrepresented; satellite sequences may not replicate beyond their level in 2C cells. Hence the pathways of endoreplication in three cell types, salivary gland, nurse, and follicle cells, share basic features of DNA replication, and differ primarily in the extent of association of the duplicated chromatids.

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.

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a "doubling of DNA" mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a "doubling of DNA" mechanism. In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined three regions of under-represented euchromatic DNA sequences (histone, Ubx, and 11 A), for their possible correlation with euchromatic constrictions in polytene chromosomes of Drosophila melanogaster. Cloned sequences were hybridized to filters and to chromosomes prepared for light microscopy. Under-represented sequences hybridized to DNA within constrictions and in ectopic fibers. In contrast, adjacent sequences that were fully endoreplicated in the Ubx and 11 A regions in polytene cells hybridized to sites just adjacent to their respective constrictions. For one region (Ubx), sequences under-represented in salivary gland cells were fully endoreplicated in fat body cells. For this particular region, the morphology of the polytene chromosomes differs between these two cell types in that the specific constriction is absent at this region in fat body polytene chromosomes, thus strengthening the correlation between under-representation and chromosome constrictions. Although all three sequences are in regions that have been classified by others as "intercalary heterochromatin," we detect no common functional or sequence organizational feature for these examples of under-represented DNA. We suggest that the lower efficiencies of the replication origins, or special regions of termination at these sites, are the primary cause of the under-replication, and that this under-replication is sufficient to confer the properties of intercalary heterochromatin.

We have used an electron microscopic analysis to define and to characterize active transcription units of Drosophila melanogaster. The lengths and spacings of nascent ribonuclear protein (RNP) fibers were determined on embryonic chromatin that was spread using techniques introduced by Miller and Beatty (1969). The data are consistent with the occurrence of specific sites of transcription initiation and termination. We apply the term transcription unit (TU) to a chromatin region bounded by these control sites. Two classes of TUs are active in Drosophila melanogaster embryonic cells--those synthesizing ribosomal RNA and those synthesizing non-ribosomal RNA. The classes can usually be distinguished on the basis of TU size, chromatin morphology and inferred DNA packing ratio, frequency of RNP fibers (number of fibers per mum of chromatin), and the solitary vs. tandem repeat occurrence of fiber arrays. The results indicate that non-ribosomal transcription units have lengths in accord with the expectation that DNA of each chromomere is transcribed as a unit. Some nascent fiber arrays in D. melanogaster have more complex patterns of RNP fiber lengths. We suggest that these are a consequence of cleavage of RNP fibers at specific sites during transcription. These sites of transcriptional control and the amounts of DNA between them provide a basis for further relating units of transcription to units of gene function.