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.     

Messenger-like RNA synthesis and DNA chromosomal puffs in the salivary glands of Rhynchosciara americana

RNA synthesis was studied mainly in the proximal sections of Rhynchosciara salivary glands in late fourth instar at two typical periods of development. These are characterized either by the absence or presence of the so-called “DNA puffs” in the salivary gland chromosomes. It was found that simultaneously with the appearance of the DNA puffs there is a great increase in the synthesis of all RNA species. The greatest increase was found to take place in the rate of synthesis of messenger-like RNA. Four main classes of messenger-like RNA were detected, having mobilities corresponding to 33, 23, 16, and 14 S RNA. There is a correlation between the abundance of the 16 S messenger-like RNA and the degree of opening of the B-2 DNA puff. This species might therefore be transcribed from this puff.     

Ecdysteroid titers and changes in chromosomal activity in the salivary glands of Rhynchosciara americana

Titers of ecdysone and 20-OH ecdysone were measured separately in both hemolymph and salivary glands of metamorphosing Rhynchosciara larvae. Gland titers were consistently higher than hemolymph titers. Although 20-OH ecdysone was the most prominent form of the hormone, measurable quantities of ecdysone were also observed throughout development in both tissues. Changes in salivary gland replication and puffing activity could be correlated with changes in gland 20-OH ecdysone titers. This was true for both developmentally changing RNA puffs and DNA puffs, which occur during the prepupal period. The DNA puffs are tied to the final DNA replication cycle, and both this cycle and the period of amplification can be correlated with increases in gland 20-OH ecdysone content. Various aspects and possible interpretations of the above correlations are discussed.This work is dedicated to the memory of Prof. Hans D. Berendes     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

Synthesis and secretion of salivary gland proteins in Drosophila gibberosa during larval and prepupal development

Summary The late larvae of Drosophila gibberosa Patterson and Mainland choose different pupariation sites than the larvae of Drosophila melanogaster Meigen. Since the larvae of D. gibberosa do not attach themselves to the substratum, the salivary glands contain only a small amount of the glue proteins before pupariation. Proteins comprising the salivary gland secretions of late larvae of these two species were compared and found to be qualitatively quite different. Only five polypeptides with the same molecular masses were identified in both species. The rate of protein synthesis in the salivary glands of D. gibberosa continued to increase through the late larval stage and pupariation. As a consequence, the total amount of protein contained in the salivary glands also continued to increase after pupariation. To demonstrate temporal changes in protein synthesis from 48 h before pupariation to 28 h after pupariation, newly synthesized polypeptides were pulse labeled by culturing salivary glands in vitro. The patterns of polypeptide synthesis fell into four major groups depending upon whether the synthesis of a protein stopped shortly after pupariation, stopped during late pupariation, increased at pupariation, or was initiated after pupariation. Changing patterns of protein synthesis are correlated with the known changes in gene puffing during this developmental period.     

Protein synthesis in salivary glands of Drosophila hydei after experimental gene induction.

Several treatments, namely incubation at 37 degrees C, in the presence of arsenite, 2,4-dinitrophenol or vitamin B-6, or release from anaerobiosis induce the same set of puffs in the polythene chromosomes of salivary glands of Drosophila hydei. Analysis of changes in protein-synthetic patterns (as determined by radioautography of sodium dodecyl sulphate-gel electrophoretograms of extracts from [35S]methionine-labelled salivary glands) showed that concomitant with puff induction by these various treatments the same six strongly labelled polypeptide bands appeared. The amount of radioactive label in these peptides accounted for 25% of the total incorporation of [35S]methionine, except during incubation at 37 degrees C when it accounted for about 50%. The rate of synthesis of these peptides was maximal 1 h after the start of the puff-inducing treatment. The rate of decay of the rate of synthesis showed first-order kinetics both after removal of the puff-inducing stimulus or in the presence of actinomycin, with a half-life of approx. 4h.     

Developmental puffing patterns in salivary gland chromosomes of Rhynchosciara hollaenderi

An analysis of puff formation and regression has been carried out in 3 morphologically distinct regions of the Rhynchosciara hollaenderi salivary gland during mid-larval through pupal development. Puffing differences among these 3 regions have been found and analysed for both RNA and DNA puffs. The presence of such differences suggests that the gland regions may also be functionally differentiated. — Developmentally specific sequences of puffs have been distinguished and correlated with morphological and physiological events which occur during the development of Rhynchosciara. The DNA puffs as well as the RNA puffs enlarge and regress at predictably specific developmental stages. The presence of particular puffing sequences in the late larval to pupal period has been compared with the occurrence of known changes in the developmental ecdysone titre for Rhynchosciara. Certain aspects of this developmental picture appear to fit the ecdysone-stimulated puffing model for Drosophila, but other aspects indicate that the Drosophila-based model may not be completely applicable to Rhynchosciara.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Exposure of Drosophila melanogaster larvae to high temperature for short periods of time results in marked changes in the puffing patterns of salivary gland chromosomes. Temperature shock induces puffing at 9 specific loci; this pattern of induced puffs shows little developmental specificity and is similar in three strains of D. melanogaster (including the mutant lethal giant-larvae) and in D. simulans. Temperature shock also (i) retards the regression of some developmentally specific puffs and (ii) results in the regression of all other puffs normal to development. The effect of temperature treatment is similar in vivo and after in vitro treatment of salivary glands. The in vitro response is not sensitive to cycloheximide. A similar pattern of induced puffs to that found after temperature treatment is found during recovery of larvae from anoxia, but additional puffs are induced after anoxia. The size and duration of activity of the induced puffs is dependent upon the magnitude of the treatment.     

A dual role of 20-hydroxyecdysone in the control of protein synthesis related to DNA puff activity in the anterior region of Bradysia hygida (Diptera, Sciaridae) salivary gland

During the last 30 h of the larval stage, the salivary glands of Bradysia hygida show the amplification of some genes, resulting in the formation of two successive groups of DNA puffs, which direct the synthesis of two different sets of polypeptides. Incubation of anterior (S1) salivary gland regions, at age E7, beginning of first group of DNA puffs activity, in culture medium for 2 to 10 h results in a decrease in the synthesis of the polypeptides characteristic of this period. However, during subsequent incubation (from E7 to E7+12 h-24 h), when the second group of DNA puffs is active, S1 regions were able to synthesize some polypeptides characteristic of this period. The role of 20-OH ecdysone was studied, in vitro and in vivo, during these two periods of protein synthesis in S1 regions. The presence of the hormone was shown to be necessary to maintain, in vitro, the synthesis of the first set of polypeptides and was strongly inhibitory, in vitro and in vivo, to the synthesis of the second set of polypeptides. Thus, it is likely that the activity of the two distinct groups of DNA puffs is under opposite 20-OH-ecdysone control mechanisms.     

Mapping of the pattern of DNA replication in polytene chromosome from Chironomus thummi using monoclonal anti-bromodeoxyuridine antibodies

We present results from a nonautoradiographic study of DNA replication in polytene chromosomes from dipteran larvae. Monoclonal antibodies with specificity for 5-bromodeoxyuridine (BrdUrd) were used to localize by indirect immunofluorescence the sites of BrdUrd incorporation and to follow the dynamics of DNA synthesis in salivary gland cells of 4th instar Chironomus thummi larvae. This technique presents numerous advantages over autoradiographic procedures and allows mapping of DNA synthesis patterns at the level of resolution of one chromosomal band. Several replication patterns were observed, classified according to characteristic features, and tentatively assigned to specific periods of the S-phase. In early S-phase, DNA synthesis is first detectable in puffs and interbands, later in bands. Most chromosomal bands appear to initiate DNA synthesis synchronously; however, in bands within centromeric and heterochromatic regions the start of synthesis is delayed. At mid S-phase, all the bands show uniform staining. Subsequent staining patterns are increasingly differential with the bands displaying characteristic fluorescence intensities. As replication progresses through the late S-phase period, the chromosomes show a decreasing number of fluorescent bands. The last bands to terminate replication are located in centromeric and heterochromatic DNA-rich regions and a few bands of low DNA content in region IIAa-c.     

Replication in Drosophila chromosomes

Prolongation of larval life in Drosophila melanogaster, by growing wild type larvae at lower temperature, or in animals carrying the X-linked mutation giant is known to result in a greater proportion of nuclei in salivary glands showing the highest level of polyteny. We have examined by autoradiography the patterns of ³H-thymidine incorporation during 10 min or 1 min pulses in salivary gland polytene chromosomes of older giant larvae and of wild type late third instar larvae of D. melanogaster grown since hatching either at 24 ° C or at 10 ° C. The various patterns of labelling and their relative frequencies are generally similar in glands from the warm-(24 ° C) or cold (10 ° C)-reared wild type larvae, except the interband (IB) labelling patterns which are very frequent in the later group but rare in the former. The IB type labelled nuclei in cold-reared wild type larvae show labelling ranging from only a few puffs/interbands labelled to nearly all puffs/interbands labelled. In warm-reared wild type larvae, very low labelled IB patterns are not seen. In older giant larvae, the ³H-thymidine labelling patterns are in most respects similar to those seen in cold-reared wild type larvae. In 1 min pulsed preparations from all larvae, the IB patterns are relatively more frequent than in corresponding 10 min pulsed preparations. No nuclei with the continuous (2C or 3C) type of labelling pattern, with all bands and interbands/puffs labelled, were seen in 1 min pulsed preparations from cold-reared wild type or in giant larvae, and only a few nuclei in 1 min pulsed preparations from warm-reared wild type larvae exhibited the 2C labelling pattern. Analysis of silver grain density on specific late replicating sites in late discontinuous (1D) type labelled nuclei suggests that the rate of DNA synthesis per chromosomal site is not different at the two developmental temperatures. It is suggested that correlated with the prolongation of larval life under cold-rearing conditions or in giant larvae, the polytene replication cycles are also prolonged. It is further suggested that the polytene S-period in these larvae is longer due to a considerable asynchrony in the initiation and termination of replication of different sites during a replication cycle.     

Change in the concentration and intensity of synthesis of RNA in cell cultures of Chironomus thummi during metamorphosis

The content and intensity of incorporation of 3H-uridine in RNA of chromosomes, nucleoli and cytoplasm isolated by microsurgery from the salivary glands of larvae and prepupae of Chironomus thummi were studied following the incubation of salivary glands in the Cannon's medium with 3H-uridine. It was shown that during metamorphosis the content of RNA and intensity of 3H-uridine incorporation decrease in the nucleolus and cytoplasm in a prepupa, as compared with a larva, and suffer no changes in chromosomes in spite of much larger size of many puffs in a prepupa. The patterns of RNA synthesis in the salivary glands of larvae during metamorphosis are discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The patterns of puffing activity have been studied during the late larval and prepupal stages of Drosophila melanogaster. On the major salivary gland autosomes (chromosomes 2 and 3) 108 loci form puffs at some time during these developmental stages. The timing and pattern of activity of 83 of these puffs is found to be strictly dependent upon the age of the animals. Two major peaks in puffing activity occur. The first of these is at the time of puparium formation and the second in 8 hr. old prepupae. Both of these puffing peaks precede a moult by 4 hrs. 30 puffs are active before or at the time of both of these two moults. However, the sequence of appearance and regression of many of this group of puffs is different at the prepupal moult than at the pupal moult. 12 puffs occur only before or at the time of the prepupal moult and 13 puffs only before or at the time of the pupal moult. The functional significance of these periods of puffing activity is discussed and it is concluded that one function of this genetic activity in the salivary glands of metamorphosing Drosophila is the production of substances to be utilised during the histogenesis of the adult tissues.     

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.     

Ecdysone-regulated chromosome puffing in the salivary glands of the Mediterranean fruit fly, Ceratitis capitata.

The effect of ecdysone on the puffing activity of the polytene chromosomes of Ceratitis capitata has been studied in organ cultures of late-larval salivary glands. Culture of glands from 120-h-old larvae (puff stage 1) in the presence of ecdysone resulted in the initiation of the late-larval puffing cycle that is normally observed in 145-h-old larvae (puff stage 4). During a 7-h period in the presence of ecdysone, the puffing patterns of most loci resembled the in vivo patterns observed in the period between puff stages 4 and 10, indicating that the first puffing cycle can be initiated by the hormone and proceed almost to completion, in vitro. Culture of salivary glands in the presence of ecdysone and a protein-synthesis inhibitor, as well as ecdysone withdrawal and readdition experiments, indicated that most of the ecdysone-regulated puffs could be categorized into three classes: (i) the puffs that were suppressed immediately by ecdysone, even in the absence of protein synthesis; (ii) the puffs that were induced directly by ecdysone; and (iii) the puffs that were induced indirectly by ecdysone, that is, they were induced after a lag period of a few hours and required protein synthesis for their induction.     

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.     

The role of the hormone ecdysterone in the control of the activity of the Balbiani rings and the other puffs of Drosophila auraria

The activity of the Balbiani rings and the other puffs of Drosophila auraria salivary gland chromosomes from various stages of development was studied in vitro in the presence or the absence of various concentrations of the hormone ecdysterone. It was found that of 81 sites affected by these conditions, 69 (including the BRs) exhibit changes during normal development as well, while the remaining 12 change only under culture conditions. The results indicate that the normal profiles of certain puffs (and the BRs) are approximated more closely by the lower concentrations of the hormone and it is suggested that such low concentrations are necessary to induce the normal course of events in vivo. Various hypotheses concerning the influence of ecdysterone on the puffing patterns are discussed in view of the data presented in this report.     

The puff inducible in region 93D is responsible for the synthesis of the major “heat-shock” polypeptide in Drosophila melanogaster

In Drosophila, temperature shocks lead to the activation of definite puffs and to the appearance of definite new polypeptides. The effects of deletions and triplications of region 93D, the site of one of the largest inducible puffs, on the induced pattern of polypeptides has been studied. A direct correlation between the dose of this chromosomal region and the relative amount of the major inducible polypeptide (about 72,000 MW) has been observed. Uninduced embryos show a low basal level of synthesis of a 72,000 MW polypeptide and this synthesis sharply increases after temperature shocks. Out of a cross which segregates zygotes completely lacking the inducible site of region 93D, embryos were found which show no increased synthesis of the 72,000 MW polypeptide after temperature shocks. The inducible 72,000 MW polypeptide is distinctly larger than the larger subunit (about 66,000 MW) of glutamine synthetase 1, which is also induced by temperature shocks and whose structural gene was shown to map also in this region. Possible explanations are discussed.