X heterochromatin subdivision and cytogenetic analysis in Sciara coprophila (Diptera,Sciaridae)

Five new translocations recovered from irradiated sperm, each having a break-point in the proximal X heterochromatin, have been designated T23, T29, T32, T40, and T70. These translocations, together with Oak Ridge T1, make possible the precise localization of several genetic components including the X centromere, the controlling element, and the ribosomal cistrons. Only the data pertaining to centromere location are presented here. The ribosomal cistrons and controlling element will be dealt with separately. Full cytological details are given for each of the five translocations. The break-points on the X define three blocks of heterochromatin designated H₁, H₂, and H₃. Together they comprise the right arm of the X. H₃ is the smallest and forms the very end of the chromosome. H₁ lies immediately to the right of the centromere. In T23 and T70 the breakpoints are located between H₂ and H₃; in T29 and T32 between H₂ and H₁. In Oak Ridge T1 the break-point lies between H₁ and the centromere and in T40 between the centromere and the whole left arm of the chromosome. For the first time it has been possible to determine the exact breakpoints of the long paracentric inversion that is found on the X homologue.This series of papers is dedicated to Professor Sally Hughes-Schrader —cytologist, naturalist, scholar — who in her eighty-second year is still exulting in the wonders of chromosome behavior and still endowed with that understanding and grace which have heightened immeasurably the lives of those who have known her     

Localization of ribosomal DNA within the proximal X heterochromatin of Sciara coprophila (Diptera,Sciaridae)

By means of several reciprocal translocations in Sciara coprophila, each having a break-point in the proximal X heterochromatin, it has been possible in the salivary gland nucleus to bring about separation of specific regions of this heterochromatin and then, by means of in situ hybridization, to determine the relative number of ribosomal RNA cistrons in each. The three blocks of heterochromatin delineated by the translocation break-points have been designated H1, H2, and H3; H1 is the most proximal, lying immediately to the right of the X centromere, and H3 is the most distal, constituting the very end (right) of the chromosome. The distribution of ribosomal RNA cistrons is as follows: 10% are located in H1; 50% in H2; and 40% in H3. For the first time it has been possible to confirm by grain count our previous biochemical estimate of a 60% deletion of rRNA cistrons in the proximal heterochromatin of the X _W homologue. The grain count data also support the conclusion of our previously published cytological analysis, that the exchange points in the X heterochromatin are identical in translocations T29 and T32 (between H1 and H2), also in translocations T23 and T70 (between H2 and H3). The coincidence of break-points in the X heterochromatin is considered in relation to the chromomere make-up of the region. Also, the occurrence of ribosomal RNA cistrons in all three heterochromomeres is discussed in relation to the functional significance of chromomeres.     

Cytological and autoradiographic studies in Sciara coprophila salivary gland chromosomes

Summary Morphological and metabolic changes on the salivary chromosomes of Sciara coprophila were followed during the later half of the fourth larval instar.Cytological maps were prepared for five successive stages from mid-fourth instar to the prepupal stage. These maps, which constitute a revision of those published earlier by Crouse, summarized our cytological findings and were the basis for studies on DNA replication of these chromosomes.Similar to earlier studies in Chironomidae, differences in the puffing pattern were noted between the anterior and the posterior portions of the salivary gland. The most striking difference was noted in region 2B on chromosome III which produces a large puff only in nuclei from the anterior part of the gland. Other autosomal puffs, although present in both parts of the gland, showed constant differences in size.An increase in the number of bands from mid-fourth to late fourth instar was observed. The new bands are all of the light-staining kind.In Sciara the puffed area may include a large number of bands in addition to the bands which originated the puff. The maximal extent of puffs was determined in terms of chromosomal map regions and the number of bands subject to obliteration.In the autoradiographic experiments use was made of H³-thymidine as DNA precursor. The aim of these studies was to detect any asynchronies in the replication time of bands. In fact, marked differences in the relative rates of uptake of H³-thymidine of a number of bands in a certain proportion of chromosomes have been observed, while others showed uniform incorporation. Since these latter were found with higher frequency the period of uniform labeling must comprise a larger part of the replication cycle then the periods of localized labeling. To assess the validity and constancy of the observed patterns of unequal incorporation, a semiquantitative analysis was carried out. It showed that the bands showing localized uptake may be separated into two broad groups. In one of these groups are the centromere regions and certain chromosomal ends, which are presumably heterochromatic. The other group comprises most of the puff sites and bulbs. Since late replication is characteristic of heterochromatin, we assumed that bands of the former group (C) replicate late in the cycle, while puffs and bulbs start replication early, and the period of equal labeling is intermediate. Other intermediate labeling patterns were observed and are described.It is known that in the fourth instar from two to three DNA replications occur in the salivary gland nuclei, the last of which coincides with puffing. Several stages may be distinguished in the puffing process based on morphology and rates of isotope uptake of the puffs. The first sign of puffing is a very high rate of incorporation at puffs. It is maintained throughout this last DNA synthesis period and only declines when all other chromosomal regions have ceased to replicate. A pattern of high and exclusive uptake at the heterochromatic sites (pattern C) was never observed in this replication; instead puffs are the last regions to terminate DNA synthesis.These results are discussed in relation to several current problems, such as, asynchronous DNA replication, the problem of metabolic DNA, and the concept of the heterochromatic state.Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy, in the Faculty of Pure Science, Department of Zoology, Columbia University, New York. This work has been supported by U.S. Public Health Training Grant No. 2Tl-GM-216-05; partial support has been received also from Grants GB 42 and G-14043 from the National Science Foundation to Dr. H. V. Crouse.     

Genome size and determination of DNA content of the X chromosomes, autosomes, and germ line-limited chromosomes of Sciara coprophila

The unique chromosome biology of the fungus fly Sciara coprophila has fascinated investigators for over 80 years. Male meiosis exhibits a monopolar spindle, nonrandom segregation of imprinted chromosomes and nondisjunction of the X chromosome. The unusual mechanism of sex determination requires selective elimination of X chromosomes in embryogenesis. Supernumerary (L) chromosomes are also eliminated from the soma during early cleavage divisions. Distinctive DNA puffs on the larval salivary gland chromosomes are sites of DNA amplification. As a foundation for future genome studies to explore these many unusual phenomena, we have used DNA-Feulgen cytophotometry to determine genome size from hemocyte nuclei of male (X0) and female (XX) larvae and adults. The DNA content of the X chromosome is approximately 0.05 pg DNA and the autosomal complement is approximately 0.45 pg DNA. Measurements of DNA levels for individual sperm from adults showed that the DNA contribution of the germ line-limited (L) chromosomes constitutes as much as 35% of the DNA of the male gamete. A parallel study using Sciara ocellaris, a related species lacking L chromosomes, confirmed the presence of two X chromosomes in the sperm of this species.     

Studies on the fine structure of puffs in Sciara coprophila

Fine structure of RNA and DNA puffs of Sciara coprophila was studied during late developmental stages of the fourth larval instar. In RNA puffs the predominant structure seen seems to be a diffuse, lampbrush-like thread or threads sectioned in a variety of planes. The thread is composed of filamentous and granular material. Three types of RNA puffs, each with a slightly different morphology, are found. In their development DNA puffs pass through a precise sequence of stages, each with its distinct morphologic and metabolic characteristics. At the initial and final stages, when much of the puff chromatin is in the compacted state, DNA puffs resemble condensed chromosomal bands. In contrast, at stages when most chromatin is diffuse, DNA puffs share many structural characteristics of RNA puffs. Most of the expanded puff area is permeated by lampbrush-like threads composed of fibrils and granules. RNA and DNA puffs were compared with respect to granule size and distribution by means of electron micrographs of known magnification. The results of the statistical analysis show that: 1) The coefficient of variation (C.V.) of the method of measurement falls between 5 and 7%. 2) There is a fluctuation in granule sizes within each puff with a C.V. of 24–26%. 3) The average granule diameter is 238 Å for DNA puffs and 310 Å for RNA puffs; the difference is statistically significant. 4) The variation in mean granule size in a sample of DNA puffs is rather small (C.V. 12%), while the variation in granule size between different RNA puffs is somewhat larger (C.V. 20%). 5) The relative spread of granule sizes in DNA puffs is more restricted than that in RNA puffs. It is evident then that, on the average, DNA puff granules are smaller and more uniform than granules found in RNA puffs.     

The structure and maturation of the spermatozoa of Sciara coprophila

The axial filament of Sciara coprophila does not conform to the usual 9 + 2 filament pattern but consists, rather, of as many as 76 pairs of filaments which decrease in number from the anterior to the posterior region of the sperm. It is first seen at the base of the head in the shape of an indented oval. The axial filament varies in configuration along the remaining length of the sperm as one whorl or two connected whorls of filament pairs. The other structures of the sperm revealed by the light and electron microscopes are a homogeneous, dense, spear-shaped nucleus, a row of spherical dense bodies in the middle piece enclosed by the axial filament and of unknown nature and function and a single mitochondrial derivative. The mitochondrial nebenkern derivative consists of a large electron transparent region bordered by cristae and a smaller paracrystalline region located adjacent to the axial filament. The derivative arises as paracrystalline material in a medial nuclear indentation. The electron transparent material is first seen at the anterior end of the middle piece. Unlike other known insect sperm, but reminiscent of sperm capacitation in mammals, sperm maturation is completed in the spermathecae of Sciara 7 to 9 hours after insemination. It consists of the acquisition of sperm motility and elimination of the electron transparent region of the mitochondrial nebenkern derivative. The electron microscope reveals in mature sperm that the axial filament doublets have changed configuration and consist of a single whorl which encloses the paracrystalline rod. The process by which the major portion of the nebenkern derivative is eliminated occurs in four identifiable stages. Since sperm maturation does not appear to be intrinsically controlled, factors in the spermathecal fluid may play a role in its completion.     

The organization of DNA in the mitotic and polytene chromosomes of Sciara coprophila

The organization of DNA in the mitotic metaphase and polytene chromosomes of the fungus gnat, Sciara coprophila, has been studied using base-specific DNA ligands, including anti-nucleoside antibodies. The DNA of metaphase and polytene chromosomes reacts with AT-specific probes (quinacrine, DAPI, Hoechst 33258 and anti-adenosine) and to a somewhat lesser extent with GC-specific probes (mithramycin, chromomycin A₃ and anticytidine). In virtually every band of the polytene chromosomes chromomycin A₃ fluorescence is almost totally quenched by counterstaining with the AT-specific ligand methyl green. This indicates that GC base pairs in most bands are closely interspersed with AT base pairs. The only exceptions are band IV-8A3 and the nucleolus organizer on the X. In contrast, quinacrine and DAPI fluorescence in every band is only slightly quenched by counterstaining with the GC-specific ligand actinomycin D. Thus, each band contains a moderate proportion of AT-rich DNA sequences with few interspersed GC base pairs. — The C-bands in mitotic and polytene chromosomes can be visualized by Giemsa staining after differential extraction of DNA and those in polytene chromosomes by the use of base-specific fluorochromes or antibodies without prior extraction of DNA. C-bands are located in the centromeric region of every chromosome, and the telomeric region of some. The C-bands in the polytene chromosomes contain AT-rich DNA sequences without closely interspered GC base pairs and lack relatively GC-rich sequences. However, one C-band in the centromeric region of chromosome IV contains relatively GC-rich sequences with closely interspersed AT base pairs. — C-bands make up less than 1% of polytene chromosomes compared to nearly 20% of mitotic metaphase chromosomes. The C-bands in polytene chromosomes are detectable with AT-specific or GC-specific probes while those in metaphase chromosomes are not. Thus, during polytenization there is selective replication of highly AT-rich and relatively GC-rich sequences and underreplication of the remainder of the DNA sequences in the constitutive heterochromatin.     

Molecular characterization of DNA puff II/9A genes in Sciara coprophila

A cDNA clone, pSDII/9, that hybridizes in situ to ecdysone-regulated DNA puff II/9A in Sciara coprophila was used as a probe to isolate a Sciara genomic clone. lambda pSDII/9, which contains a 14.7 x 10(3) base-pair DNA insert. The full-length cDNA insert was sequenced and mapped to gene II/9-1 on the genomic clone. A second gene (II/9-2), transcribed in the same direction as II/9-1, was also mapped to lambda pSDII/9, and its nucleic acid sequence was found to be 85% similar to that of gene II/9-1. An RNase protection assay demonstrates that gene II/9-1 contains a single intron that also exists in gene II/9-2 according to sequencing analysis and primer extensions of RNA encoded by this gene. Computer analyses of the deduced amino acid sequences of genes II/9-1 and II/9-2 indicate that the two DNA puff-encoded proteins are mostly alpha-helical coiled-coils. The 5'-flanking sequences of both genes contain regions that are similar to other ecdysone-regulated genes from Drosophila melanogaster.     

Comparative cytogenetic analysis of Cestrum (Solanaceae) reveals different trends in heterochromatin and rDNA sites distribution

Species of Cestrum L. (Solanaceae) exhibit large variability in the accumulation of repetitive DNA, although their species possess a stable diploid number with 2n = 16. In this study, we used chromosome banding and fluorescence in situ hybridization (FISH) to characterize the karyotypes and populations of two species, Cestrum nocturnum L. and C. mariquitense Kunth. We also performed a karyotype comparison using 16 idiograms, of which 4 were developed in this study and 12 were obtained from the literature. Cestrum nocturnum displayed more bands than C. mariquitense, but the latter exhibited greater interpopulational variation in the band patterns. There was a tendency for large bands to be located at intercalary/terminal regions and for small bands to be located at intermediate/proximal regions. The idiogram comparison revealed a large variation in the amount, distribution, and size of heterochromatic bands. FISH with rDNA probes revealed stability in the number and location of 5S sites, while 45S was more variable in size and number of sites. Although 45S rDNA always appeared in the subterminal regions, this DNA family exhibited a mobility among chromosome pairs. These data highlight the dynamic of repetitive DNA families in these genomes, as well as the contribution for intra- and interspecific karyotype differentiation in Cestrum.     

Nuclear division and migration during early embryogenesis of Bradysia tritici coquillet (syn. Sciara ocellaris) (diptera : Sciaridae)

Nuclear division and migration of cleavage nuclei in the embryos of Bradysia tritici (Diptera : Sciaridae) have been studied by light microscopy and nuclear staining. There are 8 cleavage cycles up to the syncytial blastoderm stage (4.5 hr), and during the 11th cycle cellularization begins (6.5 hr). The first 3 divisions take about 30 min each. During the 5th and 6th cycles, the maximum rate of division is reached (12 min/cycle at 22°C). After pole cell formation, the duration of the following mitotic cycles increases progressively. During nuclear migration, the presumptive germ line nuclei reach the egg cortex first, followed by anterior somatic nuclei and finally, posterior somatic nuclei reach the egg cortex. Possibly as a result of this region-specific nuclear migration, nuclear divisions become parasynchronous after 3 hr of embryogenesis (4th cycle). Several mitotic cycles later, between the 8th and 10th cycle in different embryos, X-chromosome elimination in somatic nuclei begins at the anterior egg pole and progresses in anteroposterior direction. Our observations suggest that the observed region-specific differences may be due to the activity of localized factors in the egg that control migration and nuclear cycle of the somatic nuclei.     

Two distinct intervening sequences in different ribosomal DNA repeat units of Sciara coprophila.

We have prepared a partial gene library of sheared DNA from the fungus fly, Sciara coprophila, by dA-T tailing and insertion into pBR322. Two ribosomal DNA clones which differ from the usual ribosomal DNA organization in this organism were studied in detail. Clone pBc 1L-1 has an intervening sequence of 1.4 kb, and clone pBc 6D-6 has an intervening sequence of 0.9 kb. These intervening sequences occur in about the same position in 28S rDNA, but do not appear to share sequence homology with one another. Previously we found that 90% of Sciara ribosomal DNA is homogenous and lacks an intervening sequence, and our present data explains the size heterogeneity found in most of the remaining 10%. We have found no evidence of size heterogeneity in the nontranscribed spacer.     

The effect of ecdysterone on nonhistone proteins in salivary gland chromosomes of Sciara coprophila.

Synthesis and transport of proteins to the cell nucleus during puff induction was studied in S. coprophila. Changes in grain distribution along chromosomes (L-methionine [35S] incorporation into protein) were correlated with puffs induced by ecdysterone in vitro; A pattern of specific labelling at the sites of incipient puffs was noted within 2 h after the addition of the hormone, i.e. grains on the chromosomes were in clusters, characteristic for this time point and not seen in the controls (where only non-specific labeling was noted 0-4 h). Characteristic chromosomal puffs appeared between 3-4 h after the addition of ecdysterone. It was concluded that during ecdysterone-induced puff formation in salivary gland chromosomes, proteins which had been previously synthesized were selectively transported from the cytoplasm to specific sites on the chromosomes.     

Isolation and characterization of the ecdysone receptor and its heterodimeric partner ultraspiracle through development in Sciara coprophila

Regulation of DNA replication is critical, and loss of control can lead to DNA amplification. Naturally occurring, developmentally regulated DNA amplification occurs in the DNA puffs of the late larval salivary gland giant polytene chromosomes in the fungus fly, Sciara coprophila. The steroid hormone ecdysone induces DNA amplification in Sciara, and the amplification origin of DNA puff II/9A contains a putative binding site for the ecdysone receptor (EcR). We report here the isolation, cloning, and characterizing of two ecdysone receptor isoforms in Sciara (ScEcR-A and ScEcR-B) and the heterodimeric partner, ultraspiracle (ScUSP). ScEcR-A is the predominant isoform in larval tissues and ScEcR-B in adult tissues, contrary to the pattern in Drosophila. Moreover, ScEcR-A is produced at amplification but is absent just prior. We discuss these results in relation to the model of ecdysone regulation of DNA amplification.