Das Puffmuster der Borstenapparat-Chromosomen vonSarcophaga barbata

The chromosome complement ofSarcophaga barbata polytene scutellar trichogen and tormogen cells is described and the puffing sequences of chromosome IV were analysed from day 5 to day 17 of pupariation, i. e. from the beginning up to the end of bristle and socket formation at 21°C. There are 5 normal polytene chromosomes and a complex of fibrillar and granular heterochromatin in the giant cell nuclei. It is supposed that the heterochromatic masses represent the underreplicaetd sex-chromosomes. During a 13 day period of development 105 puffs appear in the trichogen cell chromosome IV respective 102 puffs in the tormogen cell chromosome IV. The puffing patterns of these two sister cells show many similarities. However, according to the differences in development, morphology and function of bristle and socket, there are also specific differences in the puffing patterns of the trichogen and tormogen cell. Preliminary observation suggest that the hormone bursicon induces some new puffs in the tormogen cell chromosomes of freshly emerged adults.     

Puffing activities and binding of ecdysteroid to polytene chromosomes of Drosophila melanogaster

Salivary glands of third instar Drosophila melanogaster larvae were incubated in vitro in the presence of 5 x 10(-6) M 20-hydroxy-ecdysone. Steroid hormone was localized on the polytene chromosomes of the salivary gland by a combination of photoaffinity-labeling and indirect immunofluorescence microscopy. Steroid hormone binding to chromosomal loci and their puffing activity was correlated for the larval/prepupal puffing cycle characterized by puff stages 1-10. In general, there was a good correlation between the sequential and temporal puffing activity induced by 20-hydroxy-ecdysone and the binding of ecdysteroid hormone to these puffs. Ecdysteroid hormone was detected at intermolt, and at early and late puffs with two notable exceptions. Ecdysteroid was not detected at the two well-studied puffs at 23E and at 25AC, the former being an early puff, which is activated in the presence of 20-hydroxy-ecdysone, and the latter being an intermolt puff, which regresses more rapidly in the presence of hormone. Ecdysteroid hormone was present at puffs as long as the respective puff was active. Also, it apparently accumulated at late puff sites after induction. Since ecdysteroid binding to chromosomal loci is temporal as well as sequential during the larval/prepupal puffing cycle, additional factors besides steroid hormone are necessary for sequentially regulating puffing and concomitant gene activity during development from larvae to prepupae.     

The effect of prolonged exposure to heat on the activity of salivary gland polytene chromosomes]

The influence of long-term heating on the puffing activity of polytene chromosomes in the early prepupa salivary glands was investigated. The activity of puffs was estimated by two criteria: size and frequency. The rearing of insects at a temperature of 29 degrees resulted in puff changes: the activity of some puffs increased or depressed, some puffs were inhibited, other puffs were induced newly. The differential response of each chromosome was observed. A possible mechanism of the effect of heating on the puff activity of polytene chromosomes is discussed.     

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.     

Experimental induction of gene activity in the salivary gland chromosomes of Trichosia pubescens (Diptera: Sciaridae)

During the course of experiments with larvae of Trichosia pubescens, we have unexpectedly found that diethyl ether or chloroform anesthesia induces a large puff in a specific band in the polytene chromosomes of the salivary glands. This puff develops a few minutes after the treatment, attaining its maximum size after 60-100 min, and regresses completely 200 min after its activation. Through autoradiography, an intense incorporation of RNA precursors into that puff was observed. A few other smaller puffs are also induced by the treatment. The treatment with diethyl ether or chloroform does not induce puffing in the polytene cells of malpighian tubules and of midgut.     

DNA-puffing patterns in the salivary glands of Trichosia pubescens (Diptera-Sciaridae)

The puffing pattern in polytene chromosomes from salivary glands of fourth-instar larvae of Trichosia pubescens was studied. It was found that the puffing pattern is rather constant during most of larval life but changes continuously in a precise sequential order during the period preceding pupation. During this period, characterized by drastic changes in the puffing pattern, amplification of specific genes and expansion of the DNA-puffs occur. The pattern of protein synthesis in the salivary gland has also been studied by means of SDS-polyacrylamide gel electrophoresis and fluorography. It changes drastically and continuously during the phases preceding pupation. These changes in protein synthesis could be correlated with the changes in the puffing pattern, essentially with the activity of the DNA-puffs.     

The action of mitomycin C on the bristle-forming apparatus ofPhormia regina

Summary Mitomycin C, a known inhibitor of DNA synthesis, was injected into white prepupae ofPhormia regina, Adults which developed from these prepupae showed alterations of the bristle pattern, loss of whole bristle organs, and the formation of bristles without sockets or sockets without bristle shafts. Dose-dependence was found for all modifications. For the abdominal microchaetae, the period of maximum sensitivity to the drug began at 16 h after puparium formation, that is well after all of the macrochaetae and most of the microchaetae of the thorax and the head had grown insensitive. Bristle forming trichogen and tormogen cells developed high degrees of polyteny with distinctly banded chromosomes. Photometric determination of the amount of Feulgen-DNA per nucleus led to estimations of DNA classes ranging from 256C to 2048 C. DNA contents of nuclei from Mitomycin C treated animals were significantly lower during the actual growth of the bristle apparatus, but reached approximately the same level as the controls prior to the time of emergence. Cytological investigations proved that doses of Mitomycin C which yielded bristle organs either without sockets or without shafts do not affect the differential division of the bristle mother cell. Polytene chromosomes damaged by Mitomycin C displayed a diffuse and irregular banding pattern. Possible modes of action of Mitomycin C on replicating polytene chromosomes are discussed.     

An overview of gene activity in the fat body of Drosophila gibberosa

In the larval fat body of Drosophila gibberosa, polytene chromosome structure and activity exhibit cytological differences from chromosomes of midgut and salivary glands. These differences include long-persisting puffs, transient puffs and long-persisting band modulations. Some early ecdysteroid-induced puffs are present in all three organs but few late puffs are present in the fat body. Comparative studies reveal, therefore, that late larval-early pupal puffing is enhanced in salivary glands relative to gut, fat body and Malpighian tubules. After the fat body breaks up in the prepupa, the rate of programmed cell death and the corresponding slow decline of chromosomal activity also differ from cell to cell and from other organs.by M.L. Pardue     

The proteins of polytene chromosomes of Drosophila hydei

It is reported that chromatin can be prepared from highly purified polytene nuclei from the salivary glands of third instar larvae of Drosophila hydei; such chromatin differs from that of diploid nuclei mainly by deficiencies in certain nonhistone chromosomal proteins. It is suggested that these proteins are important components of constitutive heterochromatin, which is severely underrepresented in polytene chromosomes. Chromosome morphology, including the pattern of induced puffs, is maintained throughout the mass isolation of glands and sucrose gradient purification of nuclei, as indicated by studies on temperature-shocked and control larvae. No significant alteration in the chromosomal proteins following puff induction by heat shock could be detected on analysis of the isolated protein fractions by disc gel electrophoresis. More sensitive techniques must be developed to study the apparent rearrangement or accumulation of protein at puff sites, and to elucidate the role of this protein in gene activation.     

Die puff-Musterfolgen der Speicheldrüsenchromosomen aus wanderlarven und vorpuppen von Drosophila melanogaster in vitro

Late larval salivary glands of D. melanogaster of an exactly defined developmental stage (VP 0, i.e. prepupae ot later than 15 min after formation of the puparium) are cultured under sterile conditions in three standard media for insect tissue culture and in Ringer solution. In chromosomes II and III, variations in puff number and size are the same in vivo and in vitro, and almost all changes in puffing pattern are very similar to those appearing in normal development. They are the same in the four media. No additional puff is ever induced due to the medium. By contrast, salivary gland chromosomes from larvae of the late third instar before pupation do show different alterations in vitro than in vivo. This points to a threshold in the course of the puffing pattern between puff stage 8/9 and 10/11. The appearance of a substance causing prepupal changes in puffing is strictly correlated with the formation of the pupanium and the beginning of the intermoult phase in the prepupa. Comparing the results of the experiments it can be stated that the new control system is not based solely on the absence of ecdysone, but also on the existence of another inducer. Immediately after puparium formation the control by ecdysone is still active, together with the control by the supposed inducer. Later, control by ecdysone respectively by the puffs of the ecdysone cycle is substituted by the new control system, up to the next moult. As far as the chemical nature of the puffing inducer in the intermoult phase is concerned, further investigations are necessary.     

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Developmental changes in the fine structure of the salivary gland of Trichosia pubescens (Morgante) (Diptera : Sciaridae) in relation to puffing activity

Two differentiated sections (S₁ and S₂) of the salivary gland of Trichosia pubescens (Morgante) (Diptera : Sciaridae) have been examined by electron microscopy for fine structural alterations that occur in the cell cytoplasm during larval development. Such changes have been correlated with the puffing patterns of the polytene chromsomes. During stage 1 (end of the 3rd instar to mid 4th instar), the puffing pattern and the ultrastructure of S₁ and S₂ cells are rather constant. Nevertheless, marked differences are noted when the puffs and the fine structure of the 2 sections are compared. In S₁, secretory material is concentrated and eliminated as membrane-bound granules, while in S₂, secretory granules are not detected and the elimination of secretion seems to occur continuously. At stage 2 (end of the 4th instar), the puffing pattern undergoes considerable alterations simultaneously with the appearance of many ultrastructural modifications. In S₁, the morphological aspect of the secretory granules is altered, while in S₂ a decline in the secretory activity is detected. At stage 3 (older 4th-instar larvae), most of DNA puffs are active, there being no striking differences in the puffing pattern between S₁ and S₂. This stage is marked by the onset of gland histolysis, with the appearance of an intense autophagic activity of lysosomes in S₁ and S₂. As histolysis progresses during stage 4 (prepupae and early pupae) the activity of the polytene chromosomes decreases; most of the cells present a large number of autophagic vacuoles and an increasing disorganization of the cytoplasm, leading to the final lysis of the gland.     

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.