Comparative study of the function of polytene chromosomes in laboratory stocks of Drosophila melanogaster and the l(3)tl mutant (lethal tumorous larvae)

A study of the puffing pattern of the salivary gland autosomes of D. melanogaster was performed through the last 24 hours of larval development and 0-hour prepupae. Since both prominent and small puffs were taken into account, the total puff number amounted to 275. Of these, 116 are almost constant in size during the 24 hours observation period, 106 increase in size or appear before pupation. 37 puffs are active in 96 hour larvae and disappear or decrease sharply in size by 115–118 hours. 12 biphasic puffs have been found with higher activity in 96 hour larvae and 0-hour prepupae and lower activity by 115–118 hours. Three extremely irregular puffs have been detected in chromosome 4. The data obtained evidence that a larger number of D. melanogaster polytene chromosome loci are active during larval development than it has been thought earlier. It has also been shown that only 38% of autosomal puffs change before the beginning of metamorphosis. The functional significance of small puffs and strain specificity of puffs are discussed.     

The polytene chromosomes in the fat body nuclei ofDrosophila melanogaster

A photo-map of the polytene chromosomes of fat body nuclei from third instar larvae ofDrosophila melanogaster has been constructed and keyed to the revised salivary gland maps of Bridges (1938), Bridges and Bridges (1939) and Bridges (1941a, b, 1942). Apparent variations in banding pattern are discussed in the light of current studies on polytene chromosome structure and function.     

Polymorphisms in the genomic distribution of copia-like elements in related laboratory stocks ofDrosophila melanogaster

Summary The genomic distributions of the copia, 297, 412, mdg 1, and B 104 transposable elements have been compared by the Southern technique among two Oregon R and four Canton SDrosophila laboratory lines that have been maintained separately for defined periods of a few years. The heterogeneity of the autoradiographic patterns suggests that multiple transposition events have occurred during the time of separation. The hypothesis that transposition could be induced by, variations of environmental parameters is discussed.Presented at the FEBS Symposium on Genome Organization and Evolution, held in Crete, Greece, September 1–5, 1986     

Development of cuticular patterns in the legs of a cell lethal mutant ofDrosophila melanogaster

Summary The development of cuticular patterns in the legs ofDrosophila melanogaster was studied in the temperature-sensitive cell autonomous lethal mutant1 (1)ts726 by treating animals with heat pulses of two days' duration at different developmental stages, in order to find out whether or not models which account for regulation of imaginal discs in the late third instar also hold for earlier developmental periods. Eight kinds of phenotypes were found, each of which occurred only after heat pulses that started at particular time: (1) complete and incomplete mirror image duplications of mesothoracic legs: early second instar; (2) homoeotic transformation to wing hinge in mesothoracic legs: early second instar; (3) prothoracic leg fusions: early second instar; (4) hypertrophied sex combs: early third instar; (5) outgrowths: early third instar; (6) sex comb teeth on second tarsal segment: early third instar; (7) reversed bristle polarity in intersegmental membrane gaps: early third instar; (8) deleted individual bristles: middle of third instar. These phenotypes were compared with patterns predicted by two models that have been devised to account for regeneration data: the polar coordinate model, and the gradient-of-morphogenetic-potential model. Some of the data (especially the finding of circumferentially incomplete partial duplicates) are more readily predicted by the polar coordinate model, although neither model can be ruled out. Phenotypes (6) and (7) can be accounted for by postulating a tandemly repeated positional signal corresponding to tarsal segmentation. The homoeotic transformation may be due to a transdetermination event occurring in situ during regulative growth following cell death. Since deletion of individual sex comb teeth leads to altered sex comb rotation, it is suggested that adjacent sex comb tooth cells interact during rotation.Address until September 1978: Institute of Molecular Biology, Billrothstraße 11, 5020 Salzburg, Austria     

In situ patterns of nuclear replication in brain ganglia ofl(2) gl 4 mutant larvae ofDrosophila melanogaster

The1(2) gl ⁴ mutation (a deletion mutation of a recessive oncogene) ofDwsophila melanogaster causes neuroblastoma in the optic centres of brain of late third instar larvae. We have studied thein situ patterns of DNA synthesis in these brains by immunocytochemical detection of cells incorporating 5-bromodeoxyuridine. It was seen that1 (2)gl ⁴ brains from younger 3rd instar larvae had fewer replicating cells than in wild type larvae of comparable age but in brain ganglia of olderl (2) gl ⁴ larvae the number of replicating cells was much higher. The spatial distribution of replicating cells in optic lobes of brain ganglia of1 (2) gl ⁴ larvae was disturbed from early 3rd instar stage, much before the tumourous growth was morphologically detectable. The stereotyped pattern of asymmetrical cell divisions of the neuroblasts and their progeny cells was also not seen in1 (2) gl ⁴ brain ganglia. Therefore, it appears that thel (2)gl ⁴ product has an important role early in development to determine the temporal and spatial patterns of neuroblast cell division in developing brains.     

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.     

Proteins of the brain and body wall in larvae ofDrosophila melanogaster

Proteins of the brain and body wall cells of third instar larvae ofDrosophila melano-gaster have been examined by two-dimensional gel electrophoresis. Out of over 600 [³⁵ S ]-labelled peptide spots seen in brain or body wall extracts, 517 were common to both; 61 spots were unique to brain and 66 unique to muscle. Glycoproteins were identified by soaking the gels in radioactive iodinated Concanavalin-A. Forty four Con-A binding glycoproteins were identifiable in the brain and 41 in the muscle extracts. Out of these, 8 glycoproteins of the brain and 8 of muscles appear to be tissue-specific.     

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.     

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.     

DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues ofDrosophila melanogaster

Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformedDrosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatichsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatichsp26 gene. In contrast, the heterochromatichsp26 transgenes are present in approximately the same copy number as the endogenous euchromatichsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV.     

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.     

Electron microscope mapping of the pericentric and intercalary heterochromatic regions of the polytene chromosomes of the mutant Suppressor of underreplication in Drosophila melanogaster

Breaks and ectopic contacts in the heterochromatic regions of Drosophila melanogaster polytene chromosomes are the manifestations of the cytological effects of DNA underreplication. Their appearance makes these regions difficult to map. The Su(UR)ES gene, which controls the phenomenon, has been described recently. Mutation of this locus gives rise to new blocks of material in the pericentric heterochromatic regions and causes the disappearance of breaks and ectopic contacts in the intercalary heterochromatic regions, thereby making the banding pattern distinct and providing better opportunities for mapping of the heterochromatic regions in polytene chromosomes. Here, we present the results of an electron microscope study of the heterochromatic regions. In the wild-type salivary glands, the pericentric regions correspond to the beta-heterochromatin and do not show the banding pattern. The most conspicuous cytological effect of the Su(UR)ES mutation is the formation of a large banded chromosome fragment comprising at least 25 bands at the site where the 3L and 3R proximal arms connect. In the other pericentric regions, 20CF, 40BF and 41BC, 15, 12 and 9 new bands were revealed, respectively. A large block of densely packed material appears in the most proximal part of the fourth chromosome. An electron microscope analysis of 26 polytene chromosome regions showing the characteristic features of intercalary heterochromatin was also performed. Suppression of DNA underreplication in the mutant transforms the bands with weak spots into large single bands.     

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.     

Phenotypes of lethal alleles of the recessive temperature sensitive paralytic mutantstambh A ofDrosophila melanogaster suggest its neurogenic function

The mutantstambhA ¹ (2–56.8) ofDrosophila melanogaster was identified as a reversible temperature sensitive adult and larval paralytic. We have (i) isolated and analysed phenotypes of one new homozygous viable paralytic allele and two recessive unconditional embryonic lethal alleles ofstmA and (ii) studied the interaction of the viable paralytic alleles with ts paralytic mutantsnap ^ts1 (2–55.2) andpara ^ts1 (1–53.9). The homozygous viable paralytic allelesstmA ² andstmA ¹ are semi dominant neomorphs. The lethal allelesstmA ¹² andstmA ⁷ appear to be amorphs. Unhatched embryos expressing lethalstmA alleles showed hypotrophy of the anterior dorsal cuticle overlying the brain with a concomitant hypertrophy of the anterior dorsal neurogenic region (the brain). The ventral cuticle was poorly differentiated, and the ventral nerve chord showed mild hypertrophy and poor organisation. The epidermal cells in 12–13 h old embryos did not show the normal palisade layer arrangement. These phenotypes are similar to mutant phenotypes of the neurogenic class of genes whose wild type functions are necessary for intercellular communication. The allelesstmA ¹ andstmA ² do not appear to interact with the paralytic mutantsnap ^ts1 orpara ^ts1 in double mutant combinations. On the basis of our results it is proposed thatstmA may belong to the neurogenic class of genes.     

Structural features of 3C6/C7 interband chromatin organization in Drosophila melanogaster polytene chromosomes

Mechanisms of chromatin decompaction in interbands of Drosophila polytene chromosomes have been studied. Using the example of interband 3C6/C7 of the X chromosome, we investigated the ability of different DNA segments to form an interband in a new genetic environment. We applied site-specific FLP recombination between two transposons with FRT-sites that allows introducing the DNA fragments from the interband 3C6/C7 into pICon(dv) transposon located in cytologically well-characterized 84F region of chromosome 3 followed by electron microscopic analysis of changes in the region caused by insertion of the DNA fragments into the transposon. It was shown that the insertion of a 276-bp DNA fragment from the 3C6/C7 region into the pICon(dv) transposon leads to the formation of a new interband between two thin bands represented by the transposon material. This DNA fragment is the known minimal sequence that is necessary and sufficient for interband generation. In addition, the sequence containing three copies repeated in tandem of 0.9 kb DNA from the interband 3C6/C7, including the 276-bp fragment, were integrated in the transposon. The presence of introduced DNA fragments did not change the morphology of the resulting interband. It was shown that the sites of DNase I hypersensitivity were saved in transposon sequences introduced into a new genetic environment. The data obtained allow analysis to be started of specific factors (proteins, DNA motifs, etc.) that determine the formation of decompacted chromatin in a certain interband region and chromomeric organization of interphase chromosomes in Drosophila as a whole.