Distribution patterns of three subfractions of drosophila nonhistone chromosomal proteins: possible correlations with gene activity.

The distribution of three molecular weight subfractions of the Drosophila nonhistone chromosomal proteins (NHC proteins) has been studied using an immunofluorescent technique (Silver and Elgin, 1976). In all three cases, the fluorescence distribution patterns obtained are distinct and reproducible. The results imply that different NHC protein components have different distributions along the polytene chromosomes. A highly selective pattern is obtained using antiserum against subfraction ?; puffs (loci highly active in RNA synthesis) and many nonpuffed chromomeres which are known to puff at other times during the third larval instar or prepupal stages are brightly fluorescent. New RNA synthesis can be induced at 87A, 87B-C1 and other chromomeres by heat shock treatment; these loci, previously stained at low levels, are subsequently stained brightly using the ? serum. The staining of the heat shock puffs appears to be superimposed upon the prior ? pattern. The results suggest that a change in chromosomal structure, as indicated by staining using the ? serum, is associated with gene activity as indicated by puffing. This different chromosomal structure may be the consequence of either a redistribution of a ? antigenic determinant [a new association of specific protein(s) with the active sites] or a change in chromatin configuration [making the ? antigenic determinant(s) newly available to the antibody probe].     

Immunoelectron microscopic localization of RNA polymerase B on isolated polytene chromosomes of Chironomus tentans

RNA polymerase B (or II) was localized by immunoelectron microscopy in ultrathin sections of polytene chromosomes isolated from larval salivary glands of Chironomus tentans. The enzyme was found at decondensed sites (puffs and interbands), whereas no detectable RNA polymerase B was present in condensed loci (bands). Within each of the large puffs the highest enzyme concentration was observed wherever the chromatin was in the most decondensed state. Otherwise the enzyme appeared homogeneously distributed within puffs and interbands. This immunoelectron microscopic study, along with the recently published immunofluorescent and autoradiographic analysis of isolated Chironomus chromosomes (Sass, 1982) unequivocally demonstrates that RNA polymerase B is present in most, if not all interbands.     

Monoclonal antibody directed against RNA polymerase II of Drosophila melanogaster

Summary Monoclonal antibodies were raised against purified RNA polymerase II (or B) from Drosophila melanogaster. The antibody produced by one hybridoma cell clone was found to be directed against the two large subunits of the enzyme. The absence of antibodies directed against proteins possibly contaminating the antigens used for immunization allowed us to identify RNA polymerase unequivocally in interbands and puffs of polytene chromosomes. Within a single heat shock puff (87C1) RNA polymerase was found to be clustered in two separate areas suggesting two distint regions of RNA polymerase activity in this puff.Abbreviations FITC fluorescein isothiocyanate - PAGE polyacrylamide gel electrophoresis - PBS phosphate buffered saline - SDS sodium dodecyl sulfate - Enzyme DNA-dependent RNA polymerase or nucleotide-triphosphate - RNA nucleotidyltransferase (EC 2.7.7.6)     

Isolation and distribution of a Drosophila protein preferentially associated with inactive regions of the genome

The distribution patterns of chromosomal proteins from Drosophila can be observed by immunofluorescent staining of the polytene chromosomes from larval salivary glands. We have purified a non-histone chromosomal protein of M_r=69 000 molecular weight which has a high affinity for DNA with little sequence specificity. Immunofluorescent staining indicates that this protein is preferentially associated with the inactive portions of the genome, including the centric heterochromatin and the condensed bands within the euchromatic arms of the chromosomes. Observation of both the heat shock loci 87A and 87C and the developmentally regulated loci 74EF and 75B shows an inverse correlation between immunofluorescent staining for the M_r=69 000 protein and for RNA polymerase. The presence of this protein appears to be correlated with the packaging of the chromatin in an inactive form.     

RNA Polymerase B (or II) in heat induced puffs of Drosophila polytene chromosomes

Using indirect immunofluorescence visualization techniques we investigated the distribution of RNA polymerase B (or II) and histone H1 at heat shock puff loci in Drosophila melanogaster polytene chromosomes at different times during and after heat shock. After heat treatments of from 5 to 45 min, the heat shock puff displayed intense fluorescence when stained for RNA polymerase B, but relatively little fluorescence when stained for histone H1. Returning heat shocked larvae to room temperature resulted in the appearance of a distinctive pattern of RNA polymerase-associated fluorescence in the heat shock puff at 87C, presumably reflecting events associated with the inactivation and regression of this puff. Large differences observed in the apparent RNA polymerase B content of puffs of similar size suggest that the interaction of RNA polymerase B with chromosomal loci does not depend on simply the state of condensation or decondensation of the chromatin.     

A protein released by DNAase I digestion of drosophila nuclei is preferentially associated with puffs.

Antisera have been produced against five molecular weight subfractions of the Drosophila proteins readily extracted from nuclei following limited DNAase I digestion. Immunofluorescence staining techniques were used to assess the distributions of these proteins in the polytene chromosomes of Drosophila. In three cases, the antigens were widely distributed; in one case, the antigens appeared to be slightly more concentrated at active loci; and in one case, the antigens were strongly concentrated at a defined set of loci, including puffs and most of the loci which are active (puffed) at some time during third instar larval and prepupal development. The latter distribution pattern differs from that of RNA polymerase. Nonhistone chromosomal proteins of this type may have a key role in establishing and/or maintaining the altered chromatin structure characteristic of the active state.     

Activity of the endogenous RNA polymerase on fixed polytene chromosomes.

Fixed polytene chromosomes can serve as templates for RNA synthesis in situ, using the endogenous chromosomal DNA-dependent RNA polymerase. Labelling is mainly localized in band regions. However, radioactivity can also be found in interbands and puffs similar to that which occurs in vivo. It is also found by this technique that the nucleolar RNA polymerase appears to be active in these preparations and requires Mg²⁺ for activity. Since the pattern of the RNA transcribed in situ with the DNA-dependent RNA polymerase from E. coli of native chromosomes differs from that with endogenous RNA polymerase and resembles the one obtained with heat-treated chromosomes, it is suggested that the polymerase from E. coli does not act specifically on eukaryotic chromosomes.     

Tritiated-histidine incorporation into Drosophila salivary chromosomes

An autoradiographic study of H³-histidine incorporation into nonhistone protein of explanted larval salivary gland chromosomes of D. virilis showed patterns of incorporation that were dependent upon the stage of larval development. The sequence of changes in the development of several puffs in a specific chromosomal region was followed using the appearance of pigment in the anterior spiracles as a means of larval staging. H³-histidine incorporation into these puffs in prepupae occurred as the puffs were regressing in size and protein staining. Acid extraction of histone and nucleic acid failed to alter the character of the autographs; presumably a non-histone protein is involved in the H³-histidine incorporation. Other puff sites in the same prepupal chromosomes showed various patterns of isotopic amino acid incorporation indicating that the pattern reported for a specific region may not be true for all puff sites.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.