Oscar L. Miller

Ultrastructural patterns of RNA synthesis during early embryogenesis of Drosophila melanogaster

Chromatin was obtained from Drosophila melanogaster during early embryogenesis and examined by transmission electron microscopy. Nuclear contents spread at progressive stages of syncytial development show a low level of only non-nuclear template activity, and very few RNP fibril gradients extending over 2mum in length are observed. At the cellular blastoderm stage, newly activated nucleolar genes appear during the early portion of the first true cell cycle. Variation in the lengths of incomplete rRNP gradients indicates that the activation of each rRNA gene is independently controlled. All rRNA loci, whether having complete or incomplete gradients, exhibit high densities of nascent transcripts per unit length, suggesting that the rate of chromatin transcription, rather than the RNA polymarase I pool size, limits rRNA synthesis on individual genes. No more than half the rRNA genes are derepressed at this stage indicating that total rRNA synthesis is regulated by the number of genes activated. Non-nucleolar RNP fibril gradients covering up to 8 mum of genome are also first observed at the cellular blastoderm stage. Most of these gradients are differentiated from the short gradient first seen during syncytial growth by a lower density of transcribing RNA polymerases.

Novel amplification and transcriptional activity of chorion genes in Drosophila melanogaster follicle cells

Single-copy chorion genes coding for egg shell proteins are amplified in the follicle cells of Drosophila melanogaster egg chambers. Chromatin spreads of appropriately staged follicle cells reveal complex, multi-forked chromosomal structures in which one chromatin strand branches into two, which themselves branch out, and so on. In one micrograph, 13 strands originating from a single strand were observed. These structures can account for the maximal amplification occurring in the center of the domain, where the major chorion genes are located, and the decreasing amplification of flanking sequences to both sides. The amplification, high transcriptional rate, stage-specific expression, and correlation with known molecular sizes have allowed the putative identification of the single-copy, major chorion genes on the X chromosome and on chromosome III.

Correlation of hnRNP structure and nascent transcript cleavage

Using electron microscopy of spread chromatin, we have observed nonnucleolar transcription units from Drosophila melanogaster and Calliphora erythrocephala that display specific cleavage of nascent transcripts. We have quantitatively analyzed 20 of these relatively long transcription units. The primary RNP structure of homologous transcripts is nonrandom with respect to both RNA sequence and the cleavage event. In general, released RNA fragments have a smooth fibrillar RNP morphology (approximately 50 A wide) and retained segments have a thicker particulate morphology (approximately 250 A diameter). A characteristic secondary structure formation also accompanies cleavage--that is, RNP fibril loops form by association of noncontiguous transcript sequences that correspond to the terminal regions of the segment to be released. RNP particles form at the loop base sequences prior to their association and apparently coalesce upon loop formation. These loops, and thus the released segments, range in length from 1 and 25 kb on the examples we have analyzed. Cleavage of nascent hnRNA transcripts appears to be a fairly common event in these organisms and occurs within 0.3-3 min after transcription of the cleavage site.

Post-replicative nonribosomal transcription units in D. melanogaster embryos

We have analyzed transcription units from D. melanogaster embryos in which most cells were in either the late S or the G2 phase of a synchronous cell cycle. Much of the chromatin of these embryos was observed in the form of parallel strands which appear to be newly replicated sister chromatids (McKnight and Miller, 1977). Lateral ribonucleoprotein (RNP) fibers were commonly observed attached to chromatin strands and are believed to be nascent transcripts. Very similar lateral fiber arrays were found on sister chromatid strands. Such paired, post-replicative RNP fiber arrays offer a unique opportunity to compare the transcription patterns on two copies of the same transcription unit. Analyses of paired transcription units yield the following conclusions. Transcription initiation and termination sites are the same for both copies of each transcription unit. Each transcription unit has a characteristic and constant RNP fiber frequency, and the fiber frequency is identical for both copies of the transcription unit. Active transcription units can have internal, fiber-free gaps which may result from interruptions in initiation of transcription. Transcription units differing in polarity and fiber frequency can occur in immediate juxtaposition. The relevance of these findings to eucaryotic transcriptional control mechanisms are discussed.

The 5′→3′ polarity of the xenopus ribosomal RNA precursor molecule

Abstract Evidence is presented that in Xenopus laevis the 18S rRNA sequence is proximal to the 5′ end of the rRNA precursor molecule and the 28S sequence is proximal to the 3′ end. This assignment was made by digesting amplified ribosomal gene transcription complexes with EcoRI restriction endonuclease and spreading the cleaved transcription complexes for electron microscopy. From the known location of the EcoRI cutting sites within the transcribed region and the length of nascent chains on the cleaved transcription complexes, it was possible to make an unambiguous assignment of polarity.

Visualization of the major late r strand transcription unit of adenovirus serotype 2

Abstract Using chromatin spreading for electron microscopy, we have visualized the major late r strand transcription unit of adenovirus serotype 2 in a transcriptionally active state. Adenovirus 2 genomes were identified in the dispersed nuclear contents of infected HeLa cells by their discrete length (8 to 12 μm), relatively smooth deoxyribonucleoprotein configuration, and occurrence as both Y-form and linear form replicative intermediates. At 16 hours after infection, near the time of maximal synthesis of late messenger RNA, any one genome was likely to exhibit either several or no nascent transcripts. The percentage of adenovirus 2 genomes utilized as transcriptional templates appeared low and was not limited to non-replicating molecules. Analysis of individual transcribing viral genomes revealed an average of 3.9 transcripts per adenovirus 2 genome (ranging between 1 and 15; n = 51) and, in general, single gradients of fibril length. Linear regression analyses on 20 genomes with four or more transcripts yielded an average extrapolated promoter site at adenovirus map unit 16.6, very near the previously determined promoter for the late r strand transcription unit of 16.4. Both individual genomes and a constructed “composite genome” exhibited increasing gradients of fibril length from the promoter to the distal end of the genome, suggesting that only one large transcription unit was active, and that transcription was completed before discernible RNA cleavage events. Viral transcripts exhibited a high degree of ribonucleoprotein packaging and a secondary structure suggesting intramolecular interactions. The low transcript density on the viral genomes was similar to that seen on host heterogeneous nuclear RNA transcription units. A calculation is presented that indicates that the observed frequency of transcription is sufficient to account for late viral mRNA production.

Visualization of transcriptional activity during xenopus iaevis oogenesis

Amphibian oogenesis has been divided into six discrete stages on the basis of morphological changes which occur during oocyte development (Dumont 1972). Stage 3, the maximum “lampbrush” stage, is characterized by highly extended chromosomes and by lateral loops along the chromosome axes that are known to be active sites of RNA synthesis. Dumont stage 4 Xenopus laevis oocytes undergo chromosome condensation and increase in size to become mature stage 6 oocytes which are 1200–1300 µm in diameter and marked by an equatorial white band separating the animal and vegetal hemispheres. Previous cytological studies have indicated that the lampbrush loops of mature oocytes are contracted, suggesting that a decrease in synthetic activity occurs at late stages of oogenesis (Dumont 1972; Muller 1974). However, recent biochemical evidence (Anderson and Smith 1978) indicates that stage 6 X. laevis oocytes exhibit rates of nonribosomal RNA synthesis that are as high or higher than those of stage 3. We have reexamined transcriptional activity in stage 3 and stage 6 X. laevis oocytes using phase contrast and electron microscopy.

Ultrastructural analysis of polytene chromatin of Drosophila melanogaster reveals clusters of tightly linked co-expressed genes

Patterns of gene activity on individual chromatids of polytene chromosomes of Drosophila melanogaster white prepupae were ultrastructurally characterized by electron microscopy. The band-interband structure of salivary gland polytene chromosomes is lost when they are dispersed in a low ionic strength detergent solution. Morphologically similar, active genes in close proximity to one another were seen in dispersed white prepupal chromatin. The arrays of genes almost certainly represent sister copies of the same locus. Although lateral register between gene copies on multiple strands was not maintained, analysis of sister transcriptional units of unknown identity was achieved at the periphery of the chromatin arrays. Juxtaposed genes with divergent transcriptional polarity were prevalent. The morphology, size and transcriptional polarity of multiple copies of short, tandemly organized, RNA polymerase dense, co-expressed gene clusters is reported. One highly transcriptionally active region, designated the white prepupal locus (WPP locus), composed of a co-expressed tandem cluster of ten genes within an approximately 50 kb region was analyzed on six separate chromatids. The transcriptional map suggests that the pattern of gene activity for at least one gene within the cluster may not be identical on all homologous strands. The survey of active polytene genes provides ultrastructural correlation with previous molecular data that demonstrate tight linkage of certain developmentally co-regulated Drosophila genes. Our findings are discussed in relation to Drosophila gene organization, clustering, and regulation of gene expression.

Some ultrastructural aspects of genetic activity in eukaryotes.

SUMMARY A brief overview is given of the types of information that have been obtained by chromatin spreading methods regarding the ultrastructure and regulation of genetic units. The examples used are: ribosomal RNA genes of the spotted newt; the silk fibroin gene of Bombyx mori; chorion gene amplification in Drosophila melanogaster; RNA synthesis patterns during early embryogenesis of D. melanogaster; regulation of transcription on homologous non-ribosomal transcription units of D. melanogaster; specific nascent transcript cleavage in insects; and nascent polypeptides on insect polyribosomes. The conclusion is drawn that further innovations will be required before significant advances in the use of chromatin spreading techniques to study ultrastructural aspects of genetic activity can occur.