Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.     

Deoxyribonucleic acid synthesis in isolated polytene nuclei of Drosophila hydei

DNA synthesis has been studied in polytene nuclei isolated from larval salivary glands of Drosophila hydei. The incubation conditions employed promote maximum incorporation of TTP-H³ and retention of normal polytene chromosome morphology. The chromosome structure is sensitive to the Mg²⁺ concentration; a normal banding pattern is observed between 4 and 10 mM Mg²⁺. At the optimum pH of 7.8, incorporation continues for over an hour. All four deoxyribonucleoside triphosphates are required for maximum incorporation. The reaction is stimulated by 0.6 mmATP and strongly inhibited at higher ATP concentrations. Competition experiments demonstrate that either TDP or TTP is the effective labeled precursor. The labeled product is sensitive to DNase and has a density identical to that of nuclear DNA. Autoradiographs prepared from spread chromosomes demonstrate that discontinuous and continuous labeling patterns observed in vivo are also produced with isolated nuclei in the absence of cytoplasmic factors. Incubation of the isolated nuclei results in a low level of uniform incorporation that is superimposed on the normal autoradiographic pattern obtained after in vivo labeling. This background incorporation can be greatly increased by prior irradiation of the glands. The presence of exogenous DNA during nuclear incubation stimulates total incorporation. These observations demonstrate that the isolated nuclei possess a reserve synthetic capacity. About 20% of the isolated nuclei are inactive in DNA synthesis.This investigation was supported by PHS Research Grant No. 5 R01 GM 16298 from the National Institute of General Medical Sciences.     

Developmental changes in the major inclusion bodies of polytene nuclei from larval tissues of the blowfly,Calliphora stygia

The major ribonucleoprotein inclusion bodies of the polytene nuclei from larval ventral nephrocytes, Malpighian tubules, pericardial cells, fat body and salivary glands of Calliphora stygia show characteristic developmental patterns at the light microscope level. Three morphologically and developmentally distinct classes of nuclear inclusions occur in these tissues. In fat body and salivary gland nuclei, morphological changes in the inclusion material can be correlated with the pattern of protein synthesis during the third larval instar, and with conspicuous changes in chromosome morphology. The appearance of cytologically diverse nuclear inclusions, in an ordered tissue-specific sequence, may thus reflect differential activation of the genome.     

Hydroxyurea-induced inhibition of DNA puff development in the salivary gland chromosomes of Bradysia hygida

The injection of hydroxyurea at a critical time during the fourth larval instar inhibits the development of all DNA puffs in the salivary gland chromosomes of Bradysia hygida. RNA puff formation is not disturbed and larval development continues. The effect is explained as a result of a selective and general inhibitory action of the drug on DNA synthesis during the time when gene amplification occurs in the salivary glands. The incorporation of uridine into the chromosome regions where DNA puff development has been inhibited is sharply decreased in comparison with the incorporation into non-amplifying parts of the same chromosomes. The interpretation proposed for the cytologic observations seems to offer a better understanding of the nature of the DNA puffs.     

Structure and activity of salivary gland chromosomes of Drosophila gibberosa

In Drosophila gibberosa the maximum secretory output of the salivary glands is in the prepupa rather than in the late third-instar larva. Using salivary chromosome maps provided here we have followed puff patterns from late second-instar larvae through the time of histolysis of the salivary glands 28–32 h after pupariation and find low puff activity correlated with low secretory activity throughout much of the third larval instar. Ecdysteroid-sensitive puffs were not observed at the second larval molt but do appear prior to pupariation initiating an intense cycle of gene activity. The second cycle of ecdysteroid-induced gene activity a day later, at the time of pupation, appears somewhat damped, especially for late puffs. Salivary chromosome maps provided here may also be used to identify homologous loci in fat body, Malpighian, and midgut chromosomes.     

Autophagy and Apoptosis Coordinate Physiological Cell Death in Larval Salivary Glands of Apis mellifera (Hymenoptera: Apidae)

Larval salivary glands of bees provide a good model for the study of hormone-induced programmed cell death in Hymenoptera because they have a well-defined secretory cycle with a peak of secretory activity phase, prior to cocoon spinning, and a degenerative phase, after the cocoon spinning. Our findings demonstrate that there is a relationship between apoptosis and autophagy during physiological cell death in these larval salivary glands, that adds evidence to the hypothesis of overlap in the regulation pathways of both types of programmed cell death. Features of authophagy include cytoplasm vacuolation, acid phosphatase activity, presence of autophagic vacuoles and multi-lamellar structures, as well as a delay in the collapse of many nuclei. Features of apoptosis include bleb formation in the cytoplasm and nuclei, with release of parts of the cytoplasm into the lumen, chromatin compaction, and DNA and nucleolar fragmentation. We propose a model for programmed cell death in larval salivary glands of Apis mellifera where autophagy and apoptosis function cooperatively for a more efficient degeneration of the gland secretory cells.

Addendum to:

Programmed Cell Death in the Larval Salivary Glands of Apis mellifera (Hymenoptera, Apidae)

E.C.M. Silva-Zacarin, G.A. Tomaino, M.R. Brochetto-Braga, S.R. Taboga, R.L.M. Silva de Moraes

J Biosci 2007; 32:309-28     

Genetic and cytogenetic analysis of the olive fruit fly Bactrocera oleae (Diptera: Tephritidae)

The genetic and cytogenetic characteristics of one of the major agricultural pests, the olive fruit fly Bactrocera oleae, are presented here. The mitotic metaphase complement of this insect consists of six pairs of chromosomes including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the polytene complements of three larval tissues, the fat body, the salivary glands and the Malpighian tubules of this pest has shown (a) a total number of five long chromosomes (10 polytene arms) that correspond to the five autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes, (b) the constancy of the banding pattern of the three somatic tissues, (c) the absence of a typical chromocenter as an accumulation of heterochromatin, (d) the existence of reverse tandem duplications, and (e) the presence of toroid tips of the chromosome arms. The in situhybridization of genes or DNA sequences to the salivary gland polytene chromosomes of B. oleaeprovided molecular markers for all five autosomes and permitted the establishment of chromosomal homologies among B. olea, B. tryoniand Ceratitis capitata. The heat shock response of B. oleae, as revealed by heat-inducible puffing and protein pattern, shows a higher thermotolerance than Drosophila melanogaster.     

Evidence for De Novo rearrangements of Drosophila transposable elements induced by the passage to the cell culture

The genomic distribution and the number of elements of eleven transposon families have been compared by the Southern technique between permanent cultured cells, larval salivary glands and the brains and whole flies of an inbred Drosophila line (inb-c) from which the cells were established. In cultured cells, changes in restriction patterns consistent with various types of rearrangements such as amplification, transposition and excision of the elements of copia, 1731, 412, 297 and mdg-4 transposon families are detected whereas B 104, G and blood elements appear stable. In previous reports these rearrangements were not detected among individuals of the inb-c line or among samples of somatic tissues, or in samples spanning years of maintenance of cultured cells. Hence, we believe that they have been induced de novo during the passage to the cell culture.     

Cytogenetic and molecular aspects of position effect variegation in Drosophila melanogaster

A chromosomal region subjected to position effect variegation was analysed for possible DNA under-replication. DNA clones from the vicinity of the euheterochromatin junction and from a distance of hundreds of kilobase pairs were used as probes. Formation of compact blocks of chromatin is regarded as a characteristic feature of position effect variegation. It was shown that in T (1;2) dor ^var7 males undergoing position effect variegation clones representing the DNA nearest to the breakpoint in 2B7 hybridized normally in situ to the compact blocks, providing evidence against DNA underreplication. In females the same clones did not hybridize to the compact blocks. These variations in hybridization may be related to different degrees of compaction of chromosome regions in males and females. A correlation between the degree of underreplication and the level of cell polyteny was shown by Southern-blot hybridization of a DNA probe from the 2B region to DNA from an X/O strain carrying Dp (1;1)pn2b displaying position effect variegation and compaction in 94% of salivary gland cells. Almost complete underreplication of the DNA of this region was found in salivary gland cells (with a maximal degree of polyteny), intermediate underreplication was found in fat body cells (with an intermediate degree of polyteny), and replication was not disturbed in diploid cells of the larval cephalic complex.by W. Beermann     

Tec29 controls actin remodeling and endoreplication during invagination of the Drosophila embryonic salivary glands

Epithelial invagination is necessary for formation of many tubular organs, one of which is the Drosophila embryonic salivary gland. We show that actin reorganization and control of endocycle entry are crucial for normal invagination of the salivary placodes. Embryos mutant for Tec29, the Drosophila Tec family tyrosine kinase, showed delayed invagination of the salivary placodes. This invagination delay was partly the result of an accumulation of G-actin in the salivary placodes, indicating that Tec29 is necessary for maintaining the equilibrium between G- and F-actin during invagination of the salivary placodes. Furthermore, normal invagination of the salivary placodes appears to require the proper timing of the endocycle in these cells; Tec29 must delay DNA endoreplication in the salivary placode cells until they have invaginated into the embryo. Taken together, these results show that Tec29 regulates both the actin cytoskeleton and the cell cycle to facilitate the morphogenesis of the embryonic salivary glands. We suggest that apical constriction of the actin cytoskeleton may provide a temporal cue ensuring that endoreplication does not begin until the cells have finished invagination.     

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.     

Underreplication during polytenization?

Summary Recent cytophotometric DNA determinations and results of labeling experiments are compared with results of biochemical experiments concerning larval polytene salivary gland nuclei of Drosophila melanogaster. Recent publications (Dennhöfer 1981; 1982 a, b) demonstrate that methodological errors both in hydrolysis of the DNA before Feulgen reaction and in interpretation of the cytophotometric values give raise to the hypothesis of heterochromatic underreplication during polytenization. It is concluded also that methodological difficulties cause the absence of polytene SAT-DNA in biochemical centrifugation experiments since, because of different solubilities of eu- and heterochromatic DNA, the latter is not resolved in DNA isolation procedures from polytene nuclei.     

Replication in Drosophila chromosomes

The temporal order of replication of specific sites in polytene chromosomes from salivary glands and gastric caeca of Drosophila nasuta larvae was compared using ³H-thymidine autoradiography. Labelling of different cytological regions in segments of chromosome 2R (section 47 A to 49 C) and chromosome 3 (section 80 A to 82 C) was examined in detail in nuclei showing late S-period labelling (2 D and 1D types) in both cell types. The different labelling sites (22 on the 2R segment and 38 on the chromosome 3 segment) are cytologically similar in the two cell types. However, there are profound differences in the labelling frequencies of certain sites in polytene nuclei from salivary glands and gastric caeca during the late S-phase. This suggests that even though a comparable number of chromosomal replicating units operates in the two polytene cell types, the temporal order of completion of replication differs.     

Failure of the Amblyomma cajennense nymph to become infected by Theileria equi after feeding on acute or chronically infected horses

Tick-borne diseases in horses are caused by the intraerythrocytic protozoan parasites Theileria equi and Babesia caballi. Although T. equi is highly endemic in Latin America, the New World vector of this important parasite is controversial. The aim of this study was to test the ability of nymph Amblyomma cajennense ticks acquire infection by T. equi following feeding on infected horses. Three experiments were performed: tick acquisition of T. equi from an experimentally infected horse, tick acquisition of T. equi from naturally infected foals and tick acquisition of T. equi from a chronically infected horse. A. cajennense adults were dissected and salivary glands were collected in aliquots. Methyl green pyronin staining of the salivary glands did not show the presence of hypertrophy of acini or cell nuclei normally suggestive of Theileria spp. infection. The pools of salivary glands were negative for Theileria DNA in nested PCR assays. Histopathological analysis failed to detect sporoblast and sporozoites of T. equi in salivary gland acini. This study was not able to observe infection of the A. cajennense by T. equi.     

Homologous banding patterns in the polytene chromosomes from the larval salivary glands and ovarian nurse cells of Anopheles stephensi liston (Culicidae)

A comparison of the banding patterns of two homologous polytene chromosome arms from the larval salivary gland and ovarian nurse cell complement of Anopheles stephensi is presented. The homologous chromosomes from the somatic larval salivary glands and germ-line derived ovarian nurse cells have essentially the same band-interband organisation. An analysis of the ³H-uridine labelling patterns of a small chromosome segment from the two tissues indicates that germ-line polytene chromosomes are not radically different from somatic polytene chromosomes in their patterns of gene expression.     

Interchromosomal asynchrony of DNA replication in polytene chromosomes of Drosophila pseudoobscura

Analysis of ³H-thymidine autoradiograms of late third instar larval salivary glands of Drosophila pseudoobscura revealed a unique example of asynchrony of replication in the autosome complement. The two autosomal arms, 2 and 3, show similar labeling pattern during the initial phases, DD to 3C, and thereafter, the chromosome 3 has fewer labeled sites than chromosome 2 until the most terminal pattern, 1D. Detailed sitewise analysis of ³H-thymidine labeling shows that while nearly 54% of the sites examined in chromosome 2 have a labeling frequency greater than 50%, only 13% of all sites in chromosome 3 have labeling frequency at that range. The number of labeled sites on chromosome 3 plotted against that on chromosome 2 shows a hyperbolic profile rather than a linear relationship. The silver grain ratio of the 2nd to 3rd increases from 1.5 to 3.1 through different stages of the cycle. These results suggest that both chromosomes start replication simultaneously but the third chromosome appears to complete the replication earlier than the second. These data open up the possibility of separate control mechanisms for the initiation and termination of DNA replication in polytene chromosomes.This paper is dedicated to the memory of the late Prof. H. D. Berendes.     

Ecdysterone responsive functions in the mutantl(1)su(f) ts67g ofDrosophila melanogaster

Summary Transferring the temperature sensitive mutantl(1)su(f) ^ts67g from 25° C to 30° C before or early in the third larval instar blocks the increase in the ecdysterone titer that normally occurs at the end of the larval period. Feeding exogenous ecdysterone to these hormone-deficient larvae results in the formation of pseudopupae. The mutant was used to study ecdysterone-inducible functions in late larval salivary glands by preparing three animal samples with different hormone titers: the titer was low in one sample because of an earlier temperature shift, high in a second sample because the larvae were subsequently transferred to ecdysterone-supplemented food, and also high in a third sample that was kept at 25°C, providing a control for normal development. The effect of the different hormone conditions was studied by³⁵S-methionine labeling of the salivary gland proteins during the larval to prepupal transition and the prepupal period. The results indicate that synthesis of several of the proteins normally appearing during the transition and prepupal period is induced by exogenous ecdysterone.     

Photoinduced bonding of endogenous ecdysterone to salivary gland chromosomes of Chironomus tentans

Endogenous ecdysterone has been bonded to chromosomal loci by irradiation of Ch. tentans salivary glands. The hormone has been localized on the polytene chromosomes by indirect immunofluorescence microscopy. Hormone binding to chromosomes is stage-specific. Seven chromosomal loci could be identified which specifically bound hormone in larval salivary glands, and 21 chromosomal loci which specifically bound hormone in prepupal salivary glands. All puffs that have been described by Clever (1961) as being inducible by ecdysterone have been found to contain irreversibly bound ecdysterone in prepupal salivary gland chromosomes. A small number of puff sites in larval salivary gland chromosomes exhibited varying amounts of bound ecdysterone, (as judged by fluorescence intensity) most notably 117B and Balbiani rings 1 and 3 on chromosome IV. In addition to stage specific binding sites, there were many others showing equal binding of the hormone in both, larval and prepupal, stages of development. — Fluorescence intensities (reflecting the amount of bonded hormone) at puff sites along the tip section of the prepupal salivary gland chromosome arm IR have been computed indicating that differences between fluorescence intensities of different puffs can be expressed as multiples of a basic fluorescence intensity. Thus, the amount of fluorescence intensity (bonded hormone) in the various puffs may be quantized. — The data indicate that in Ch. tentans salivary glands ecdysterone acts, at the chromosomal level. The development of larvae into prepupae generates more puff sites and more hormone binding. This is discussed in the light of current models of hormone-receptor function.     

Characterization and chromosomal distribution of a tandemly repeated DNA sequence from the Australian sheep blowfly,Lucilia cuprina

In the course of making a Lucilia cuprina genomic DNA library, a ladder of bands was seen in partial Sau3A digests. Complete digestion reduced this ladder to predominantly monomer units of approximately 190 bp. Nine independently isolated copies of this repeat were cloned and sequenced. Only two of these isolates are identical in sequence, the most divergent being 71% homologous. This satellite DNA occurs in all three wildtype strains tested, and, for the single case examined, in the embryonic, larval, pupal, and adult DNA. It represents approximately 3%–4% of the genome. Data obtained from in situ chromosome hybridizations indicate that this sequence is concentrated around the centromeric regions of the autosomes and over most of the sex chromosomes. Labelling is much stronger in mitotic compared with polytene chromosomes showing directly that this centromeric satellite DNA is grossly under-replicated during polytenization. This under-replication is even more pronounced on the sex chromosomes compared with the autosomes.by A. Bird The EMBL accession numbers are: X57584 L.C.SAT TRS 188-1; X57585 L.C.SAT TRS 188-13; X57586 L.C.SAT TRS 188-14; X57587 L.C.SAT TRS 188-15; X57588 L.C.SAT TRS 188-19; X57589 L.C.SAT TRS 188-16; X57590 L.C.SAT TRS 188-21; X57591 L.C.SAT TRS 188-7