Observations onltl (lethal tumorous larvae) ofDrosophila melanogaster

A line ofGardner's tumorous head stock ofDrosophila melanogaster which is known to contain the inversion In (3L) Payne at varying frequencies, was found to be homozygous for another inversion, probably In (2L)t. The Payne inversion in this line is lethal when homozygous, and is balanced over a recessive lethal gene for which the symbolltl (lethal tumorous larvae) is proposed.ltl is located to the left ofRoughened on the basis of preliminary crossover tests.Homozygousltl larvae fail to pupate. They survive in the larval state for several days after pupation age. They exhibit a characteristic syndrome of abnormalities, part of which develops gradually during post-pupation age. Typical features of this syndrome are: over-abundance of blood cells, disintegration of fat-body, salivary gland, and parts of the gut, and the presence of brown or black masses (tumours). The absolute number of blood cells, and the proportion of different types of blood cells, further, the histolytic changes, in over-agedltl larvae are suggestive of changes which in normal development take place during metamorphosis. A striking feature ofltl are the melanotic pseudo-tumours. These usually float in the haemolymph; sometimes they are associated with organs. Tumour incidence is strongly influenced by genetic modifiers.A bacterial infection was found inltl. The infection is the consequence, rather than the cause, of some degenerative changes.The polytene chromosomes in salivary glands, Malpighian tubules, and parts of the gut ofltl larvae show striking abnormalities in their structure. They are shortened and thickened, and in advanced stages may assume a heterochromatine-like aspect through disintegration of the banding pattern. The changes resemble those found in conjunction with microbial infection inRhynchosciara andChironomus. In maleltl larvae the single X chromosome (in contrast to th double X in females) shows heterocyclic behaviour in that it is much shorter, wider, and fuzzier than the autosomes.Some aspects of the complex pleiotropic pattern of pathological changes inltl are discussed. Most abnormalities, including those of the chromosomes, are seen as an indirect effect, depending on physiological conditions in over-aged larvae. This is supported by observations on over-aged larvae of certain mutant types, or following treatment with 5-fluorouracil, which show similar chromosome changes. Several arguments support the view, thatltl larvae fail to pupate because of malfunction of the ring gland. Inability of the hypoderm to react is, however, not excluded as a possible explanation. Results of some transplantation experiments are included in the discussion. They show thatltl imaginal disks are capable of developing into adult organs.     

Research on the ultrastructure and function of the anal organ of the normal and lethal (l(3)tr) larvae of the Drosophila melanogaster

The anal organ of larvae of the wild type and the mutant ‘lethaltranslucida’ (l(3)tr, 3–20±0·8) of Drosophila melanogaster was studied by electron microscopy. By means of cryoscopy and microtitration the total osmotic concentration and the chloride content of the haemolymph were also determined. In addition, the effects of hypotonic and hypertonic solutions on the anal organs and the haemolymph concentration have been analysed in detail. We were especially interested in the functional significance and a possible disturbance of these anal organs in the lethal mutant.On the basis of the following characters the anal organ appears as a typical absorption organ: (a) The cuticle is distinctly thinner than that of the remaining body parts and has an enlarged surface by forming numerous porous infoldings. (b) On the cuticular surface the plasma membrane of the singlelayered epidermal cells forms a large number of folds oriented parallel to each other, resulting in a further increase of the absorption surface. (c) An extensive network of microtubules and a dense population of mitochondria, vacuoles, and vesicles in the cytoplasm suggest that an active transport process takes place in this organ.After 1 hr in a strong hypotonic solution (distilled water) the plasma membrane folds of both +/+ and l(3)tr larvae increase and penetrate deeper into the epidermal cell. The mitochondria also increase in number and are located between the apical folds. Conversely, in a hypertonic solution (1·42–11·96% NaCl) the plasma membrane folds become shorter and fewer in number. The mitochondrial density decreases. With increasing salinity and duration various unidentified bodies, degenerated mitochondria, and vesicles appear, indicating the beginning of autolysis.The osmotic concentration of 4-day-old +/+ larvae is found to be 1·02% NaCl, whereas that of l(3)tr larvae of a corresponding physiological age (5 days old) amounts to only 0·65% NaCl. Both genotypes are able to regulate the osmotic concentration in a hypotonic solution; the upper limit of salt concentration of the surrounding medium for a successful regulation is 5 per cent.The chloride concentration of +/+ larvae aged 4 days is found to be 0·19% NaCl, and that of l(3)tr larvae of a corresponding physiological age 0·14% NaCl. In a hypotonic solution both genotypes are capable of regulating the chloride concentration. However, this fails in a hypertonic medium with a concentration higher than 1% NaCl. When the anal organs are blocked by AgNO₃ impregnation, the regulatory ability breaks down completely. No distinct difference in the fine structure as well as in the regulatory achievement of the anal organs between the wild type and the l(3)tr mutant could be detected. It seems that the mutational effect does not lie primarily in a defect of the water balance.     

Elementary structures in polytene chromosomes of Drosophila melanogaster

Whole-mounted polytene chromosomes were isolated from nuclei by microdissection in 60% acetic acid and analyzed by electron microscopy. Elementary chromosome fibers in the interchromomeric regions and individual chromomeres can be distinguished in polytene chromosomes at low levels of polyteny (2⁶–2⁷ chromatids). Elementary fibers in the interbands are oriented parallel to the axis of the polytene chromosome. Their number roughly corresponds to the expected level of polyteny. These fibers have an irregular beaded structure, 100–300 Å in diameter, and there is no apparent lateral association between them in the interchromomeric regions. Most bands, in contrast, form continuous structures crossing the entire width of the chromosome. Polytene chromosomes isolated in 2% or 10% acetic acid can be reversibly dispersed in a solution for chromatin spreading. The spread chromosomes consist of long uniform deoxyribonucleoprotein (DNP) fibers with a nucleosome structure. This supports the notion that continuous DNA molecules extend through the entire length of a polytene chromosome and that the nucleosome structure exists both in bands and interbands. Analysis of the band shape and of the fibrillar pattern in the interbands emphasizes that the polytene chromosome assumes a ribbonlike structure from which the more complex three-dimensional structure of the polytene chromosome at higher levels of polyteny develops.     

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.     

Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid)

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.     

Sequence replication and banding organization in the polytene chromosomes of Drosophila melanogaster

The relative proportions of cloned DNA fragments from all known hierarchies of sequence organization in polytene and diploid chromosomes were compared. It was found that unique sequences of varying sizes and chromosomal locations are equally replicated in salivary gland chromosomes. Sequences of euchromatic polydisperse gene families are also replicated proportionately in polytene and diploid tissues. Perhaps the most significant finding is that the histone gene repeats, despite their normal banding organization, are under-replicated in the polytene chromosome of Drosophila melanogaster. However, the clustered and well-banded 5S genes are most likely equally replicated. It is therefore concluded that differential sequence replication plays no apparent role in either the assembly or morphology of a band; and likewise, the assembly of polytenic DNA into band units is not affected by either the local abundancy or arrangement of middle repetitive sequences. The likelihood that the clustered arrangement is an important factor in the selection of sequences for under-replication is discussed.     

Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes

The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.     

Identical functional organization of nonpolytene and polytene chromosomes in Drosophila melanogaster

Salivary gland polytene chromosomes demonstrate banding pattern, genetic meaning of which is an enigma for decades. Till now it is not known how to mark the band/interband borders on physical map of DNA and structures of polytene chromosomes are not characterized in molecular and genetic terms. It is not known either similar banding pattern exists in chromosomes of regular diploid mitotically dividing nonpolytene cells. Using the newly developed approach permitting to identify the interband material and localization data of interband-specific proteins from modENCODE and other genome-wide projects, we identify physical limits of bands and interbands in small cytological region 9F13-10B3 of the X chromosome in D. melanogaster, as well as characterize their general molecular features. Our results suggests that the polytene and interphase cell line chromosomes have practically the same patterns of bands and interbands reflecting, probably, the basic principle of interphase chromosome organization. Two types of bands have been described in chromosomes, early and late-replicating, which differ in many aspects of their protein and genetic content. As appeared, origin recognition complexes are located almost totally in the interbands of chromosomes.     

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.     

The interrelation of the polytene chromosomes of generative tissues in Drosophila melanogaster]

The principles of three dimensional organization of primary and secondary orders polytene chromosomes in ovarian nurse cells of Drosophila melanogaster were elucidated. Contrary to somatic tissues, no joining of chromosome arms into local chromocentre was discovered. The chromosomes are separated in the nuclear space and are attached to the nuclear envelope by the centromeric (and the XL arm--by the telomeric) sites, the arms of autosomes (especially primary polytene chromosomes) being separated in the area of attachment. Polytenized XR arm of the X chromosomes were discovered. The architecture of chromosomes discovered in ovarian nurse cells is tissue-specific and differs considerably from the organization of polytene chromosomes of somatic tissues.