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.     

Banding studies on the polytene chromosomes of Rhynchosciara hollaenderi

C-, Q-, H-, and Ag-banding have been carried out on the polytene chromosomes of Rhynchosciara hollaenderi. The results of these techniques are presented and are correlated with the molecular data which exists for Rhynchosciara polytene chromosomes. By systematic organization of both banding and molecular data, we have attempted to give as complete a picture as possible of the characteristics of the differentially banding regions. This presents an organized method of approaching mechanisms of banding in terms of structural and functional aspects of the intact chromosome. Polytene chromosomes are particularly suited for this type of analysis and with them, both developmental and evolutionary changes can be conveniently utilized for additional insights into the functions of banding regions.     

The influence of ecdysterone on gene amplification,DNA synthesis,and puff formation in the salivary gland chromosomes of Rhynchosciara hollaenderi

Many of the DNA and RNA puffing changes observed in Rhynchosciara during the prepupal period have been induced in younger larvae by injection of ecdysterone. However, the dose of hormone necessary for this induction is high, especially in the large cells of the proximal region of the gland. There are differences in the amplification and puffing response from that observed during normal development. Particular similarities and differences with possible explanations for the differences are discussed. Preceding and during the amplification which occurs at certain chromosomal regions, ecdysterone induces DNA synthesis along the entire chromosome. This induction of general DNA synthesis can occur independently of the amplification process. It appears to be similar in pattern to that occurring normally toward the end of larval life. — The normal prepupal behavior of Rhynchosciara was not induced by injection of ecdysterone into larvae of any age thus far examined.     

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.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Salivary gland X chromosome puffing patterns are described for the Oregon stock of Drosophila melanogaster and for the Berkeley stock of D. simulans. In D. melanogaster regular phase specific puffing was recorded at 21 loci in the third larval instar and subsequent prepupal stage. A comparison of the X chromosome puffing patterns of male and female larvae failed to show any qualitative differences although in the males a group of puffs were active for a longer time during development than in females. The X chromosome puffing patterns of D. simulans are similar to those described for D. melanogaster although two puffs (4F 1–4 and 7B 1–3) were active in D. simulans but not in D. melanogaster. The sex differences in puffing observed in D. melanogaster were also observed in D. simulans.     

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.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

The patterns of puffing activity in the proximal region of 2L of D. melanogaster have been reinvestigated and revised. Possible relationships between three puffs and the structural genes for alcohol dehydrogenase, dopa decarboxylase and the histones are discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

A technique for the short term organ culture of larval salivary glands of D. melanogaster is described. Cultured Puff Stage 1 glands respond to 20-OH ecdysone by initiating the cycle of puffing activity characteristic of late larval development and puparium formation. This puffing cycle involves the sequential activation of at least 125 puffs. Their response to ecdysone allows these puffs to be divided into 3 main classes: a) PS1 puffs that regress (e.g. 25AC); b) puffs activated very rapidly (within 5 min) (e.g. 23E, 74EF, 75B) and c) puffs activated only after longer periods (>4 h) (e.g. 62E, 78D, 22C, 63E and 82F). The detailed behaviour of representatives of each class is described. These data support Clever's distinction of ‘early’ and ‘late’ ecdysone responsive sites.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Patterns of puffing activity during the third larval instar and the prepupal period of two different strains of D. melanogaster (Oregon and vg6) are compared. The variation in puffing activity observed is both quantitative (involving the mean size or timing of activity of individual puffs) and qualitative. The pattern of activity of 64% of the puffs is the same in the two strains, 12% show strain differences in puff size and 19% in the time of their activity. One puff (64C) is active only in one of the strains (vg6). In genetic experiments this puff segregates normally and the puff locus has been mapped genetically to a site coincident with, or at least very close to, the cytogenetic position of the puff. In heterozygotes the puff is homozygous only when the maternal and paternal homologues are synapsed. When the homologues are asynapsed only the homologue from the vg6 parent is puffed at 64C. With the exeption of some strains closely related to vg6 no other strain of D. melanogaster has been found to possess puffing activity at 64C. In vg6/In(3LR)C165 heterozygotes 64C forms a heterozygous puff even when the homologues are synapsed. In the discussion consideration is given to the various factors that control puff size.     

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.     

Puffing activity in the salivary gland chromosomes of Rhynchosciara under experimental conditions

By using the techniques of ligation of the larvae (brain and endocrine glands extirpation) and salivary gland implantation, the hormonal dependence of the activity of certain puffs of Rhynchosciara was investigated. Our results have shown that the puffing behaviour — activation and deactivation — varies according to the developmental stage in which the larvae were ligated. When the larvae were ligated just before the drastic changes in the puffing pattern, which occur prior to pupation, these changes fail to occur. When the larvae were ligated after the onset of these changes we have observed: a) some of the puffs active at the time of the ligature regress promptly, earlier than their normal timing observed in controls; b) others remain active indefinitely and c) there are still some which regress accordingly to the normal timing.The puff B2 which behaves as those in b was double checked by means of implantation experiments. Salivary glands which had puff B2 at its maximum expansion were implanted into younger larvae and that puff also remained active in the body cavity of these larvae. Hypotheses to explain the results obtained are discussed.     

Patterns of puffing activity in the salivary gland chromosomes of Drosophila

Puffing patterns of chromosome arm 3 L of D. yakuba are compared with those of other members of the melanogaster species subgroup D. melanogaster and D. simulans. Several paracentric inversions on 3L have resulted in a considerable rearrangement of gene order in D. yakuba. However the basic sequence of changes in puffing activity which occurs during late larval and prepupal development is very similar to that of D. melanogaster and D. simulans. A fourth member of this species subgroup (D. teissieri) also has similar puffing patterns to those of D. melanogaster despite considerable chromosome evolution.     

Developmental puffing activity in the salivary gland and Malpighian tubule chromosomes ofAcricotopus lucidus (Diptera,Chironomidae)

A detailed map of the salivary gland chromosomes ofAcricotopus lucidus is presented. Differences in puffing and developmental Puffing sequences of the three salivary gland lobes were investigated from mid fourth larval instar to pupation and compared with the puffing pattern of the Malpighian tubules. The intraglandular differentiation is quite extensive; the differences in the pattern of gene activity between the anterior lobe and the main and side lobes are as great as between the salivary gland and the Malpighian tubules. In the main and side lobes all developmental puffing changes proceed synchronously whereas in the anterior lobe both asynchronous and synchronous changes occur. In the anterior lobe the asynchronous regression of BR 3 and BR 4 is followed by a characteristic sequence of activation and inactivation of puffs.     

Synthesis of satellite DNA in Rhynchosciara hollaenderi

During the latter part of the fourth instar development of Rhynchosciara larvae, DNA synthesis occurs in both germ cells and somatic cells, even though these cells do not undergo mitosis. CsCl density gradient analysis has revealed the synthesis of d(AT) satellite DNAs in the testis and in somatic tissues such as salivary gland, fat body, and gastric cecum. In these studies it has been shown that there is a tissue-specific variation in the relative proportions of synthesis of d(AT) satellite and main-band DNA in the testis during the fourth instar. The initiation of synthesis of the d(AT) satellite in the testis corresponds with the appearance and formation of the highly characteristic mitochondria which develop during the maturation of the spermatocytes. This satellite DNA has been shown to be circular and has a density of 1.681 gm/cm³ in CsCl. Synthesis of a similar circular DNA cannot be detected in somatic tissues, although these tissues do synthesize a d(AT) satellite of similar density.     

Ultrastructural aspects of histolytic processes in the salivary gland cells during metamorphic stages in Rhynchosciara hollaenderi (Diptera, Sciaridae).

The cytoplasm of Rhynchosciara hollaenderi late larval, prepupal and pupal salivary gland cells was studied at the ultrastructural level. In the second half of the 4th instar, evidence of an intensive secretory activity is visible in the form of numerous secretory granules in the apical area of the cells. At the same stage, the endoplasmic reticulum cisternae adjacent to Golgi groups are active in the transfer of vesicular elements. At later stages this activity rapidly diminishes. Before the appearance of the DNA puffs, i.e. at the end of the 4th instar, mitochondria begin to show a granular deposit and normal mitochondria decrease in number. These with the granular deposit form clusters and initiate formation of single autophagic vacuoles before the appearance of the DNA puffs. Later, at the time, when the 2B puff opens, the autophagic vacuoles appear in great number. Simultaneously with the formation of the autophagic vacuoles the presence of acid phosphatase in the Golgi vesicles and in autophagic vacuoles was shown. In the last stages investigated (late pupae) acid phosphatase is present free in the cytoplasm and at the same time disappearance of free ribosomes, pycnosis of polytene chromosomes and breakage of nuclear membranes occur. It is concluded that the histolysis of the salivary gland cells begins before the large DNA puffs appear, then it becomes very intensive and continues after these puffs undergo regression.     

Synthesis of mitochondrial DNA in spermatocytes of Rhynchosciara hollaenderi

During the mid to late 4th instar period of larval development, the mitochondria of Rhynchosciara spermatocytes undergo highly characteristic morphological changes. In late meiosis the enlarged mitochondria fuse to form a single mitochondrial element which will ultimately extend the length of the spermatid tail. Our studies have shown that synthesis of a circular DNA occurs during this period of mitochondrial “differentiation.” This DNA has a density of 1.681 g/cm³; and its synthesis cannot be detected in somatic tissues such as salivary gland, fat body, or gastric cecum. From analysis of DNA extracted from mitochondrial pellets, we have shown that the circular DNA is associated with the mitochondria. The contour length of the mitochondrial DNA is 9 μm, equivalent to a molecular weight of 18 × 10⁶. Although most metazoan mitochondrial DNAs exhibit contour lengths of approximately 5 μm (10 × 10⁵ daltons), there is no extractable 5 μm circular DNA in these spermatocytes. Therefore, we conclude that either Rhynchosciara spermatocytes possess a distinct 9 μm mitochondrial DNA or that the spermatocyte mitochondrial DNA represents dimers of 5 μm monomers.