Localization of RNA polymerase in polytene chromosomes of Drosophila melanogaster.

Abstract

RNA polymerase (RNA nucleotidyltransferase) B (or II) and histone HI of Drosophila melanogaster were localized on salivary gland polytene chromosomes using the indirect immunofluorescence technique. RNA polymerase B is present almost exclusively in puffs and interband regions, whereas histone HI is found primarily in bands. The puff at region 3C, known to be transcriptionally active in larval salivary glands, gives a bright fluorescence with antibodies against RNA polymerase B. This fluorescence disappears after exposure of the larvae to 370 for 45 min. The heat shock treatment results in a general reduction of fluorescence intensity with the appearance of brightly staining heat shock puffs. Heat-induced removal of RNA polymerase molecules from a puff does not immediately alter its morphology. We propose that an interband represents that fraction of the total number of gene copies in a band that are active, the inactive copies being present in a condensed form in the adjacent band. Large puffs would originate through the decondensation and activation of most or all gene copies in a band. Indirect immunofluorescence has become a widely applied tool for localization of not only surface antigens (1), but also intracellular components such as contractile proteins in nonmuscle cells (2). Recently, this technique has been applied to the study of the distribution of chromosomal proteins in polytene chromosomes of Drosophila (3)(4)(5). In these studies, chromatin was fractioned and either histones or nonhistone proteins, the latter being of unknown purities and functions, were used as antigens for their intrachromosomal localization. With the availability of antibodies raised against Drosophila RNA polymerase (RNA nucleotidyltransferase) B (6) we possess a probe to study the localization of a chromosomal protein of known function. Upon treatment of squash preparations from D. melanogaster salivary glands, Plagens et al. observed a distinct fluorescence pattern with the indirect immunofluorescence technique (7). They observed the a-amanitin-sensitive RNA polymerase B (also called polymerase II) not only in positions of visible puffs, but also at numerous other sites along the polytene chromosomes. Exposure of larvae to 37°resulted in a strong fluorescence at sites corresponding to the most prominent "heat shock" puffs (8). Meanwhile, a systematic study on conditions for preparing chromosomal squashes for immunofluorescence led to the observation that the postfixation with formaldehyde used previously caused shrinking of smaller puffs, yielding the impression of bands in the phase contrast pictures. This led to the original interpretation that RNA polymerase B was located also in bands (7). We studied the distribution of RNA polymerase B and of histone HI with the improved technique described here and found that RNA polymerase B occupies exclusively uncondensed regions of the chromosomes, whereas histone Hi is found primarily in condensed regions. Heat shock treatment results in a redistribution of RNA polymerase...

Locus-specific variation in phosphorylation state of RNA polymerase II in vivo: correlations with gene activity and transcript processing.

“…Previous studies using an anti-RNA Pol II serum (Jamrich et al 1977) indicated that the enzyme was located primarily in puffs and interbands, rather than in the bands that are more condensed and contain most of the DNA mass. To relate our current work to earlier experiments, we compared the anti-IIA and anti-II0 signals with a signal from DAPI, a fluorescent dye that binds to DNA and produces a pattern nearly identical to the classical banding pattern (e.g., see Fig.…”

Two closely linked transcription units within the 63B heat shock puff locus of D. melanogaster display strikingly different regulation

“…Moreover, RNA polymerase II appears to dissociate from these puffs and associates predominantly with heat shock puff sites (34,35). Chromosomal condensation (the puff to band transition) could participate in gene repression or simply be a consequence of gene repression.…”

Heat shock reduces stalled RNA polymerase II and nucleosome turnover genome-wide

“…Gene induction varies according to conditions, but this rapid transcriptional response generates 10-fold to 1000-fold induction of Hsp genes within minutes of temperature elevation (Lindquist 1986). Simultaneously, heat shock results in down-regulation of normal transcription (Jamrich et al 1977), presumably to prevent accumulation of misfolded translation products (Lindquist 1986). These rapid genome-wide transcriptional responses make the heat-shock system ideal for investigating chromatin alterations that accompany gene regulatory changes.…”