Spatial organization of chromosomes in the salivary gland nuclei of Drosophila melanogaster

Using a computer-based system for model building and analysis, three-dimensional models of 24 Drosophila melanogaster salivary gland nuclei have been constructed from optically or physically sectioned glands, allowing several generalizations about chromosome folding and packaging in these nuclei. First and most surprising, the prominent coiling of the chromosomes is strongly chiral, with right-handed gyres predominating. Second, high frequency appositions between certain loci and the nuclear envelope appear almost exclusively at positions of intercalary heterochromatin; in addition, the chromocenter is always apposed to the envelope. Third, chromosomes are invariably separated into mutually exclusive spatial domains while usually extending across the nucleus in a polarized (Rabl) orientation. Fourth, the arms of each autosome are almost always juxtaposed, but no other relative arm positions are strongly favored. Finally, despite these nonrandom structural features, each chromosome is found to fold into a wide variety of different configurations. In addition, a set of nuclei has been analyzed in which the normally aggregrated centromeric regions of the chromosomes are located far apart from one another. These nuclei have the same architectural motifs seen in normal nuclei. This implies that such characteristics as separate chromosome domains and specific chromosome-nuclear envelope contacts are largely independent of the relative placement of the different chromosomes within the nucleus.     

A reinvestigation of the nucleolus-organizing regions in the salivary gland nuclei of Drosophila melanogaster

Comparisons were made of the morphology of the proximal region of the salivary gland X-chromosome of D. melanogaster following a number of different staining procedures. Azur B was found to be the most satisfactory staining method for identification of the nucleolus. The states of eu- and heterochromatization (sensu Prokofyeva-Belgovskaya) of the bands in the proximal region—particulary striking in the mirror-image duplication of the R(1)2, ring-X, chromosome—contribute to the variability in the banding-pattern, and consequently the refractoriness of this region to cytological investigation. No nucleolus was ever found to be associated with the group of bands presumed to be the Y-chromosome.This investigation was supported in part by a U.S. Public Health Service Research Grant, GM 15009, from the Institute of General Medical Sciences of the National Institutes of Health, and in part by a grant from the Finnish National Research Council for Sciences.     

The nucleic acids of Drosophila melanogaster

1. Nucleic acids of whole Drosophila adults were prepared in good yield and substantially free from impurities by new modifications of the phenol method. 2. The average molar base compositions of the DNA (41% of guanine+cytosine) and transfer RNA (60% of guanine+cytosine) resemble those of mammalian nucleic acids; the ribosomal RNA has a DNA-like molar base composition (43% of guanine+cytosine), and it is considered that this is reflected in the lower stability of its secondary structure compared with mammalian ribosomal RNA. 3. The two main ribosomal forms were separated and average base compositions and sedimentation values determined.     

Studies on the larval salivary gland of Drosophila: I. The nucleic acids

The localization and identification of the two nucleic acids, ribonucleic and desoxyribonucleic, have been determined for the larval salivary gland of Drosophila robusta. The determinations were made by the use of basic staining, Brachet's ribonuclease method, and the Feulgen reaction.The cytological observations suggest that the gland is functional as a secretory organ up to the mid-point of the third instar. It is during this period that the cytoplasmic basophilia (RNA) is most intense. When secretion ceases there is a decided decrease in RNA concentration. These findings suggest that the RNA system is concerned primarily with secretion.     

Nucleolus-organizing regions in salivary gland chromosomes of Drosophila melanogaster

Summary The nucleolus of the salivary gland nucleus of Drosophila melanogaster is formed by nucleolus-organizing regions which exist in the heterochromatin of the sex chromosomes. This interpretation is supported by the discovery of a series of induced chromosomal alterations involving transfer of nucleolus-forming regions to euchromatic sections of the chromosomes.     

Three-dimensional organization of telomeres in the Drosophila melanogaster salivary glands nuclei

The 3D-FISH was employed to investigate the telomere topology in polytene nuclei of salivary glands of Drosophila melanogaster. The majorities of telomeres in polytene nuclei of salivary glands in Drosophila strain y(2-717) are localized in the nuclear central area and have no contacts with nuclear membrane. In females of this strain, ectopic contacts between telomeres occur at 25 % higher frequency than in males. HeT-A DNA in y(2-717alk3-2) strain, which is a derivative of y(2-717) carrying an inversion between 1D and 13C bands, is found in region 13 of X chromosome. The frequency of ectopic contacts of telomeres in y(2-717alk3-2) males is 10 % higher than that in y(2-717) strain. The number of ectopic contacts can be significantly different in independent experiments, possibly indicating the role of random factors in the contact formation.     

Rapidly labeled proteins on the salivary gland chromosomes of Drosophila melanogaster

Experiments on short-term and pulse-chase labeling of chromosome proteins of the salivary glands of Drosophila melanogaster show unique patterns of label in the vicinity of chromosome puffs. A high turnover rate is indicated for these nonhistone proteins, which appear to form a fibrous sheath around the chromosomes. Acrylamide gel analyses of the chromosomal proteins that are quickly labeled, comparing compositions at different stages of development with compositions after heat shock, show that all are different and dependent on which chromosomal puffs are active and producing messenger RNA. The necessity for a continuous and rapid interchange of protein between the nucleus and cytoplasm is indicated, and it appears that regulation of gene activity must be related to this dynamic state of protein exchange. From the technical standpoint, it has been found that scanning electron microscopy (SEM) is especially useful for observing silver grains on opaque autoradiographs. It appears also that SEM will prove useful in a variety of studies of chromosome structure.     

Cytophotometric DNA determinations and autoradiographic studies in salivary gland nuclei from larvae with different karyotypes in Drosophila melanogaster

Cytophotometric DNA determinations in Feulgen stained mitotic diploid chromosome sets of neuroblasts from larvae of Drosophila melanogaster stocks, which possess different karyotypes, show significant differences between the 4C values, caused by an additional or deficient X- and Y-chromosome depending on the karyotype. The ranges of polytenic DNA size classes are theoretically expected to be doublings of the corresponding 4C mean value of each karyotype. The extinction integral data of nuclei with completely duplicated 4C quantities exclusively fall into the range of the expected size classes. Not all data falling into the range of a size class necessarily originate from duplicated nuclei, because the limits of the DNA size classes cannot be determined by measurements, but must be estimated from the confidence limits of the corresponding 4C mean value. The validity of the mitotic 4C values of the karyotypes X/X and X/Y is tested using data from non-labeled interphase nuclei, where extinction integral data accumulate in two groups. The larger values (= G₂-nuclei) confirm the 4C values of mitotic chromosome sets, and the lower values (= G₁-nuclei) are just half of these. Extinction integrals from individual, ³H-thymidine non-incorporating polytene salivary gland nuclei accumulate in distinct, non-overlapping groups which are always complete doublings of the preceding smaller group. In each karyotype, the most frequent data of each group are in accord with the 4C doublings. The data from labeled nuclei alternate with those from unlabeled nuclei. The measured DNA values of individual polytene nuclei that did not incorporate any ³H-thymidine, demonstrate that all chromosomal DNA replicates completely during polytenization of the chromosomes in the larval salivary gland nuclei of Drosophila melanogaster. Specifically, this would mean that the heterochromatic Y-chromosome replicates as well as the partially heterochromatic X-chromosome along with the autosomes. There is no indication of underreplicating heterochromatin.     

The secretory proteins of the larval salivary gland of Drosophila melanogaster

The gene for a major salivary gland secretion protein (Sgs-1) in Drosophila melanogaster has been mapped to chromosome 2 between dp (13.0) and cl (16.5). In the late third instar larva, a puff forms in this region. This puff (25 B) regresses as the ecdysteroid concentration increases prior to puparium formation. Quantitative analysis of the secretory protein 1, showed that, when present in extra dose, region 25 B results in a significant elevation in its relative amount. This suggests that the structural gene for this protein is localized in this region and that its synthesis is directly correlated to the activity of the 25 B puff.     

The synthesis of compound bands in Drosophila melanogaster salivary gland chromosomes

Small chromosome aberrations were utilized to construct compound bands in the 3C region of Drosophila melanogaster salivary gland chromosomes. The results imply that one should expect to find multiple functions associated with single bands, especially heavy bands. It is suggested that the natural occurrence of compound bands needs to be recognized and that exceptions to the one gene: one band correspondence are expected to occur.Journal Paper No. J-9459 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 1985     

Peptidase increase accompanying growth of the larval salivary gland of Drosophila melanogaster

1. The larval salivary gland of Drosophila melanogaster offers an opportunity to study growth in a tissue in which no cell division occurs but in which the cells increase in size. 2. Measurements of alanylglycine (AG)-peptidase content have been made in three stocks of Drosophila melanogaster at different growth stages of the larval salivary gland, and have been correlated with its total nitrogen and volume. 3. During the prepupal instar, the AG-peptidase content of the gland increases parallel with total nitrogen but decreases when histolysis of the gland begins. Conversely, a benzoyl-l-arginineamide-hydrolyzing endopeptidase is not measurable until histolysis sets in. 4. In the final larval growth period of a giant mutant, there is a concomitant increase in peptidase, total nitrogen, and volume of the gland. 5. A similar association of peptidase content and total nitrogen is found in comparing glands of different sizes from the giant stock, at the time of maximal peptidase content in the prepupa. 6. The data are interpreted as evidence for an association of AG-peptidase with growth of the cells in the gland. This agrees with the earlier interpretation by Linderstr?m-Lang and Holter of data obtained from study of more complex tissues. 7. A survey of the available measurements of peptidase content in other organisms shows that wherever an increase of cell substance occurs, peptidase content increases. Conversely, peptidase remains constant where cell division is unaccompanied by an increase of cell substances. 8. The joint association of peptidases and pentosenucleic acids with protein synthesis is pointed out. 9. The possiblity is considered that peptidases may be essential parts of a unit in which coupled reactions necessary for protein synthesis occur. The r?le of the peptidases in this system is discussed. They may act either synthetically to form new peptide linkages (problematic), or hydrolytically to mobilize the necessary specific amino acids.     

Control of DNA replication and spatial distribution of defined DNA sequences in salivary gland cells of Drosophila melanogaster

In dividing cells, each sequence replicates exactly once in each S-phase, but in cells with polytene chromosomes, some sequences may replicate more than once or fail to replicate during S-phase. Because of this differential replication, the control of replication in polytene cells must have some unusual features. Dennhöfer (1982a) has recently concluded that the total DNA content of the polytene cells of Drosophila salivary glands exactly doubles in each S-phase. This observation, along with previous studies demonstrating satellite underreplication in salivary gland cells, led us to consider the hypothesis that there is a doubling of DNA mechanism for the control of DNA replication in polytene cells. With this mechanism, a doubling of DNA content, rather than the replication of each sequence, would signal the end of a cycle of DNA replication. To test this hypothesis, we have reinvestigated the replication of several sequences (satellite, ribosomal, histone and telomere) in salivary gland cells using quantitative in situ hybridization. We find that underreplication of some sequences does occur. In addition we have repeated Dennhöfer's cytophotometric and labeling studies. In contrast to Dennhöfer, we find that the total DNA contents of nonreplicating nuclei do reflect this partial replication, in accord with Rudkin's (1969) result. We conclude that DNA replication in polytene cells is controlled by modifications of the mechanism operating in dividing cells, where control is sequence autonomous, and not by a doubling of DNA mechanism. — In situ hybridization to unbroken salivary gland nuclei reveals the distribution of specific sequences. As expected, satellite, histone and 5S sequences are usually in a single cluster. This rules out the possibility that sequences known to be underreplicated in chromosomal DNA exist as extrachromosomal copies. Telomere sequences are grouped into two to six clusters, as if the chromosome ends are partially but not completely paired in salivary gland nuclei.     

Parallel changes in the puffing of polytene chromosomes and bioelectric properties of salivary gland cell nuclei in Drosophila melanogaster after heat shock

The dynamics of heat-shock puff activity and cell nuclei electrophoretic mobility in the larvae salivary glands of normal and temperature-sensitive mutant stocks of Drosophila melanogaster after heat shock (37 degrees C) were studied. The parallel changes of these characters and interlinear differences affected by ts mutation were found. Positive correlation between heat shock puff size and cell nuclei electrophoretic mobility was detected.     

Whole mount electron microscopy of the nucleolus in salivary gland cells of Drosophila melanogaster.

The nucleolus of Drosophila melanogaster salivary gland cells, examined by whole mount electron microscopy, consists of a fibrillar core region and a peripheral region containing both fibres and granules. These regions appear to correspond to the fibrillar and granular components, respectively, seen in thin sections. Most of the nucleoli were attached to the chromocenter region of the polytene chromosomes, containing the nucleolar organizer. Bundles of relatively straight chromatin fibres, 13 nm in diameter, extended from the chromocenter into the core region of the nucleolus, however it was not possible to trace the path of these chromatin fibres through the nucleolus since they were obscured within the mass of nucleolar fibres. The nucleolar fibres in both the core and peripheral regions were irregular and knobby, with a diameter of about 15 nm. In the core region, the fibres appeared to be of considerable length and were characteristically clustered together to form small interconnected masses. The fibres in the peripheral region were relatively short and some appeared to blend with amorphous, poorly-defined pools of material. Electron dense granules 15-20 nm in diameter were also associated with this amorphous substance. It is hypothesized that the formation and subsequent packaging of the 28s rRNA may be represented by a morphological transition of the peripheral fibres, via an amorphous pool-like intermediate stage, into the nucleolar granules. The results of this study indicate that whole mount electron microscopy may be a useful alternative to thin sectioning in high resolution studies of the nucleolus.