Electron microscope autoradiographic study on transcriptional activity of Drosophila melanogaster polytene chromosomes

Incorporation of ³H-uridine into three chromosome regions 21D, 100AB, 7EF showing no puffs was studied by means of EM autoradiography. These regions show rather good coincidence between EM and Bridges' revised maps. The reduction of band number observed in the EM map was mainly at the expense of “doublet” bands. — Theoretical silver grain distributions were calculated on the basis of “universal curves” (Salpeter et al., 1969, J. Cell Biol. v. 41, 1–20) on condition that either bands or interbands are linear sources of radioactivity. From these curves the resolution of EM autoradiography was deduced to be sufficient with regard to the investigated region. — The results show that in addition to the puffs peaks of silver grains occur over the interbands and diffuse bands. The lowest incorporation level is observed over the dense bands. The possibility of utilizing the data obtained for the location of RNA-synthesising regions is discussed.     

Dosage compensation of X-linked heat-shock puffs in Drophila pseudoobscura

Four major puffs are inducible by heat shock in the larval salivary gland chromosomes of D. pseudoobscura. Two of these puffs are present at 23 and 39–40 on the right arm of the X chromosome and two are present at 53 and 58 on chromosome 2. By means of in situ hybridization, residual homologies were demonstrated between the puffs at 23 in D. pseudoobscura and at 63C in D. melanogaster, and between the two chromosome 2 puffs of D. pseudoobscura and 87A and 87C of D. melanogaster. RNA synthesis was monitored as a function of ³H-uridine incorporation in the major heat-induced puffs of D. pseudoobscura and was found to be equivalent in males and females indicating dosage compensation of the two X-linked loci. The evolution of the regulatory controls of these genes is discussed.     

Amino acid induced changes of puffing patterns in Chironomus salivary gland chromosomes

Effects of amino acids on puffing in salivary gland chromosomes of Chironomus thummi are described. All twenty naturally occurring amino acids except tyrosine were tested. Of these, only tryptophan and phenylalanine proved effective. Both induced puffs at the same seven loci: Ia3/4, Ie3, If4, Ig1, Ig2, IIId1 and IIId1/2. The induction is energy dependent and requires concomitant RNA synthesis. Amino acid induced puffs incorporate ³H-uridine into acid insoluble material. At least one monovalent plus one divalent cation has to be present along with the amino acid in the incubation medium for puff induction.     

Action of hydroxyurea on chromosome physiology in Rhynchosciara angelae

Experiments have been performed to investigate the action of hydroxyurea (H.U.) on the polytene chromosomes of the salivary gland of Rhynchosciara angelae. After different times of H.U. treatment, larvae were injected with ³H-thymidine for a pulse of 10 min. DNA puffs were analysed especially in those regions where differential incorporation of thymidine occurs. H.U. progressively inhibited thymidine incorporation all over the chromosome. The maximum of inhibition occurs 9 hours after the treatment. However, after 227 hours the chromosome label was similar to that in the controls and puff 2B recovered its original size. The puff 3C showed a delay in its appearance. Our results show that H.U. inhibits temporarily the opening rate of the puff, as well as DNA synthesis. There is no reaction on RNA puffs.This work was supported by a grant from the National Institutes of Health (GM 17590-03), Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) of which one of us (G.M.M.S.) was a fellow during this research, and the Conselho Nacional de Pesquisas (CNPq).     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The autosomal salivary gland chromosome puffing patterns of Drosophila simulans are described and compared with the puffing patterns of the sibling species D. melanogaster. During the late third larval instar and the prepupal period the patterns of puffing activity of these two species are similar — approximately 50% of the puffs common to both species showing identical activities. The remaining puffs differ in their timing of activity, or in their mean sizes, or in both of these parameters. A number of puffs (14) found in D. simulans have not been regularly observed in the Oregon stock of D. melanogaster but are active in other D. melanogaster strains. One puff (46 A) of D. melanogaster was absent from D. simulans and forms a heterozygous puff in hybrids, when the homologous chromosomes are synapsed. When the homologues are asynapsed a puff at 46 A is restricted to the melanogaster homologue. The puff at 63E on chromosome arm 3L is considerably smaller in D. simulans than in D. melanogaster and this size difference is autonomous in hybrids. Other puffs not common to both species behave non-autonomously in the species hybrid, even when the homologous chromosomes are asynapsed.     

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.     

Induction of chromosome puffs in Drosophila hydei salivary glands after inhibition of RNA synthesis by α-amanitin

The effect of injection of various concentrations (4 ng to 0.5 g/larva) of -amanitin on chromosomal RNA synthesis in larval salivary glands of D. hydei was investigated at subsequent time intervals (90 min to 20 hrs) after injection. — As revealed by autoradiography, ³H-uridine incorporation into the polytene chromosomes was greatly reduced as compared with that in control larvae. In -amanitin injected larvae, new chromosome puffs could be induced by a temperature treatment. These puffs never attained a size comparable to that in the controls. The newly induced puffs did not show specific incorporation of ³H-uridine. — Puffs which were active before the toxin was applied undergo a reduction in size and incorporation of ³H-uridine.     

Hydroxyurea-induced inhibition of DNA puff development in the salivary gland chromosomes of Bradysia hygida

The injection of hydroxyurea at a critical time during the fourth larval instar inhibits the development of all DNA puffs in the salivary gland chromosomes of Bradysia hygida. RNA puff formation is not disturbed and larval development continues. The effect is explained as a result of a selective and general inhibitory action of the drug on DNA synthesis during the time when gene amplification occurs in the salivary glands. The incorporation of uridine into the chromosome regions where DNA puff development has been inhibited is sharply decreased in comparison with the incorporation into non-amplifying parts of the same chromosomes. The interpretation proposed for the cytologic observations seems to offer a better understanding of the nature of the DNA puffs.     

Local protein accumulation during gene activation

Measurements of the integrated absorbancy of naphthol yellow S binding to protein (430 nm) and Feulgen-stained DNA (550 nm) of two puff regions in Drosophila hydei polytene chromosomes revealed a significant increase in the naphthol yellow S binding capacity during the first 5 min of puff induction. The ratio of integrated absorption values at 430 and 550 nm of two chromosome regions, 2-48 C and 4-81 B were determined relative to the ratio of absorption values at 430 and 550 nm of a reference band. These determinations were carried out in a non-puffed state and at 5, 10, 30, 60 and 120 min after onset of a temperature treatment inducing puffs in these regions. The quotient of the absorption ratio of the puff region and the ratio of the reference band provides a relative measure for naphthol yellow S binding to protein. The staining reaction was absent after pronase treatment.—The relative increase in naphthol yellow S binding was most obvious during the first 5 min after onset of puff induction. The binding of naphthol yellow S was increased by a factor 1.7 for puff 2-48 C, and a factor 1.9 for puff 4-81 B. The maximum value, indicating a relative increase by a factor 1.8 in puff 2-48 C and a factor 2.2 in puff 4-81 B was attained in both puffs at 30 min after onset of puff induction.—Among staining procedures performed on sulphydryl groups, free -amino acids and indole groups of tryptophane, only a positive result with the staining reaction on the indole groups was obtained for induced puffs.—Injection of tritiated sodium acetate, methionine-H³-methyl, ethionine-H³-ethyl, C¹⁴-sodium bicarbonate, a mixture of 15 H³-labelled L-amino acids and H³-tryptophane at various time intervals prior to puff induction failed to result in a specific incorporation of any of these radioactive substances into newly induced puffs.     

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.     

A STUDY OF THE NUCLEOLAR MATERIAL IN SCIARA COPROPHILA

In the polytene chromosomes of Sciara coprophila, in addition to a nucleolus, large numbers of nucleolarlike structures or micronucleoli are formed. A detailed mapping localized the nucleolar organizer at one end of the X chromosome and revealed that approximately 18% of the bands of each chromosome are potentially capable of producing micronucleoli. Most of these sites are in regions known from a previous study to show asynchronous DNA replication: DNA puffs and certain heterochromatic regions. Micronucleoli are rarely found in association with bulbs. The RNA metabolism of the polytene chromosomes during late fourth instar was studied using radioautographic techniques. Isolated glands were incubated in tritiated uridine for 10 to 30 min, and radioautographs were made of squash preparations. Despite the wide range of variation found among different larval cultures, the following pattern was observed. Just prior to and at the beginning of DNA puff formation, a period of intense extrachromosomal nucleolar and micronucleolar RNA synthesis occurs. After maximal development of the DNA puffs, the synthesis of extrachromosomal RNA is at a low point, while incorporation into bulbs and DNA puffs remains high. With the onset of the prepupal stage, all nuclear RNA synthesis ceases.     

Comparison of homologous polytene chromosomes in Phaseolus coccineus embryo suspensor cells: morphological,autoradiographic and cytophotometric analyses

The functional behaviour of unpaired homologous polytene chromosomes (2n=22), was investigated in nuclei of Phaseolus coccineus embryo suspensor cells. Observations were carried out on the morphological level and after ³H-thymidine and ³H-uridine autoradiography. Histone and total protein contents in the chromatin were also investigated. It was shown that corresponding regions of homologous chromosomes may show different functional structures. ³H-thymidine incorporation demonstrated differences between homologues in both DNA synthesis leading to chromosome endoreduplication (polytenization) and DNA amplification (extra DNA synthesis). ³H-uridine autoradiography showed that homologous regions in a given chromosome pair may display three labeling patterns: i) both regions labeled; ii) both regions unlabeled; iii) one region labeled and the other unlabeled. These three states are found to occur in different cells of one and the same embryo suspensor. Differences between homologous chromosome regions were also found in the ratios between DNA and protein contents in their chromatin. These results, which show that the functional activity of homologous chromosomes of the same complement may greatly differ, are discussed in relation to the characteristics of the system investigated.