Localization of RNA from heat-induced polysomes at puff sites in Drosophila melanogaster.

Heat treatment of D. melanogaster tissue culture cells causes drastic changes in the pattern of protein synthesis and the size distribution of polysomes. Like the heat shock puffs on polytene chromosomes which appear while preexisting puffs regress, heat shock proteins appear on gels while the synthesis of preexisting proteins is sharply reduced, and heat-induced polysomes appear on gradients after preexisting polysomes have disappeared. Most of the poly(adenylic acid)-containing RNA isolated from high-temperature polysomes sediments in sucrose gradients and migrates in gels as a rather narrow band. This RNA is of sufficient size to code for one particular protein that is found to account for more than half of the total synthesis at high temperature. The RNA hybridizes in situ mainly at chromosome sub-division 87B, the site of the major heat shock puff.     

CONTROL OF GENE ACTIVITIES IN THE POLYTENE CHROMOSOMES OF Chironomus tentans BY ECDYSONE AND JUVENILE HORMONE

The injection of ecdysone (molting hormone) into chironomid larvae is known to result in the stimulation of specific gene activities (increased RNA synthesis). The morphological manifestation of this gene activation is the formation of a puff. Cytological and autoradiographic analysis of several regions of the salivary gland chromosomes of Chironomus tentans revealed that ecdysone also stimulates Balbiani ring 1. In contradistinction to the puffs, the Balbiani rings are tissue specific and account for a significant percentage of the cell's non-nucleolar RNA.The application of juvenile hormone results in a decreased activity of Balbiani ring 1, suggesting that the two hormones may act antagonistically. In addition, juvenile hormone induces a puff at chromosome region I-19-A and this puff becomes less active after a short-term treatment with ecdysone. These data demonstrate an effect of juvenile hormone on puffing and are the first to demonstrate activation of a Balbiani ring by ecdysone.     

Chromosome puff activity and protein synthesis in larval salivary glands of Drosophila melanogaster.

Secretion proteins from larval salivary glands of Drosophila melanogaster were analyzed with acrylamide gel electrophoresis. Four fractions were found; three showed electrophoretic variants in different wild-type stocks. Crossbreeding and cytogenetic techniques were used to localize the genes responsible for the two main fractions: The gene for fraction 3 was found to lie within a segment of the third chromosome which includes section 68C; the gene for fraction 4, Sgs-4, was found to lie within section 3C8-3D1 of the X chromosome (1 - 3-5). The puffs within these sections of the giant chromosomes are active before and during secretion synthesis and become inactive as secretion synthesis ceases. Larvae of one wild-type stock which lack protein fraction 4 do not exhibit any puffing in 3C. The relative amount of protein 4 in the salivary secretion shows a marked dependence on the dosage of the Sgs-4 gene in both duplication and deficiency genotypes. The active site within puff 3C11-12 apparently contains the structural gene for protein 4.     

Regular versus as-needed short-acting inhaled beta-agonist therapy for chronic obstructive pulmonary disease

Regular short-acting inhaled beta-agonist therapy is of uncertain benefit in patients with chronic obstructive pulmonary disease (COPD). We conducted a randomized, concealed, double-blind, placebo-controlled crossover trial in two periods, each of 3-mo duration, involving 53 patients with a smoking history of > 20 pack-years, an FEV1 of < 70% predicted, and an FEV1/VC ratio of < 0.7 after inhalation of 200 microg albuterol. All patients received regular ipratropium bromide at 20 microg per puff in 2 puffs four times daily, beclomethasone at 250 microg per puff or equivalent corticosteroid in 2 puffs twice daily, and open-label inhaled albuterol as needed. Interventional therapy consisted of regular inhaled albuterol (100 microg per puff, in 2 puffs four times daily) versus placebo. Patients used twice as much active albuterol in the regular use period (mean: 8.07 puffs of coded and 4.68 puffs of open-label medication; total: 12.75 puffs daily) than during the as-needed period (mean: 6.34 puffs of open-label albuterol daily). Despite greater beta-agonist use, patients showed similar results during treatment and control periods for all outcomes. Differences between active and placebo periods were: FEV1: -0.04 L (95% confidence interval [CI]: -0.09 to 0.01 L); slow vital capacity: 0.04 L (95% CI: -0.12 to 0.20 L); 6-min walk test distance: -3.1 m (95% CI: -16.8 to 10.5 m); and Chronic Respiratory Questionnaire scores for dyspnea: 0.02 (95% CI: -0.13 to 0.16); fatigue: -0.02 (95% CI: -0.25 to 0.20); mastery: 0.01 (95% CI: -0.20 to 0.24); and emotional function: 0.02 (95% CI: -0.20 to 0.24). We found that in patients with COPD, use of regular short-acting inhaled beta-agonists resulted in twice as much beta-agonist use without physiologic or clinical benefit as did use on an as-needed basis.     

In vitro effects of juvenile hormone on puffing in Chironomys salivary glands

Isolated salivary glands of Chironomus tentons prepupae were incubated in a synthetic culture medium containing 10^?4 M β-ecdysone and various amounts of C18-Cecropia juvenile hormone (JH). After 2 or 4 h their chromosomal puffing patterns were analyzed. Between 3.4 × 10^?7 and 3.4 × 10^?5 M, JH induces the ‘juvenile’ puff I-19-A₁. At higher concentrations (3.4 × 10^?4 M), a previous induction of this puff is reversed. The ecdysone-dependent puff I-18-C becomes progressively reduced at JH concentrations above 3.4 × 10^?6 M. Wounding of glands results in a transient, JH-independent induction and reduction, respectively, of puffs I-19-A₁ and I-18-C, and eventually, in the formation of a new puff (I-19-A₂); wounding generally diminishes the effects of JH on puffing. In the absence of ecdysone, puffs I-19-A₁ and I-19-A₂ develop spontaneously, and 3.4 × 10^?4 M JH no longer reverses puff formation in I-19-A₁. These results suggest an antagonistic interaction of JH and ecdysone with a cellular regulatory circuit involving cell membrane permeability changes.     

Parallel changes in puffing activity and patterns of protein synthesis in salivary glands of Drosophila.

The changes in protein synthesis of salivary glands of Drosophila resulting from a brief exposure at 37 degrees have been analyzed on sodium dodecyl sulfate--acrylamide gels. In D. melanogaster and D. hydei this treatment induces nine and six new puffs, respectively, in the polytene chromosomes. After 20 min treatment seven new proteins are synthesized by the glands of D. melanogaster and six by those of D. hydei as detected by [35S]methionine labeling. Other agents, e.g., recovery from anaerobiosis, induce the same puffs and the same proteins. The extent of protein induction and the degree of puff induction are related to the severity of the temperature treatment. The new proteins are detected after 10 min treatmene at 37 degrees and their synthesis is inhibited by actinomycin D. Actinomycin D added 5 min after the start of temperature treatment has little effect on subsequent protein synthesis. The induced proteins are not tissue specific. Electrophoretic differences of two proteins exist between D. melanogaster and D. simulans, encouraging attempts to map the proteins' gene loci and to test directly whether or not the puffs code for them.     

Organization of human histone genes.

We describe the isolation and initial characterization of seven independent lambda Charon 4A recombinant phages which contain human histone genomic sequences (designated lambda HHG). Restriction maps of these clones and localization of the genes coding for histones H2A, H2B, H3, and H4 are presented. The presence of histone encoding regions in the lambda HHG clones was demonstrated by several independent criteria including hybridization with specific DNA probes, hybrid selection/in vitro translation, and hybridization of lambda HHG DNAs to reserve Southern blots containing cytoplasmic RNAs from G1-, S-, and arabinofuranosylcytosine (cytosine arabinoside)-treated S-phase cells. In addition, the lambda HHG DNAs were shown to protect in vivo labeled H4 mRNAs from S1 nuclease digestion. Based on the analysis of the lambda HHG clones, human histone genes appear to be clustered in the genome. However, gene clusters do not seem to be present in identical tandem repeats. The lambda HHG clones described in this report fall into at least three distinct types of arrangement. One of these arrangements contains two coding regions for each of the histones H3 and H4. The arrangement of histone genes in the human genome, therefore, appears to be different from that in the sea urchin and Drosophila genomes in which each of the five histone-encoding regions (H1, H2A, H2B, H3, and H4) is present only once in each tandemly repeated cluster. At least one clone, lambda HHG 41, contains, in addition to the histone genes, a region that hybridizes with a cytoplasmic RNA approximately 330 nucleotides in length. This RNA is not similar in size to known histone-encoding RNAs and is present in the cytoplasm of HeLa cells predominantly in the G1 phase of the cell cycle.