Salivary Gland Polytene Chromosomes Research Articles

Chromosome variability in Chironomus acidophilus Keyl, 1960 from the Afon Goch, UK – a river subject to long-term trace metal pollution

The structure and function of the salivary gland polytene chromosomes of the larvae of the chironomid Chironomus acidophilus in a UK river subject to long-term acid (pH < 3.0) mine drainage (AMD) was examined. Two types of chromosome rearrangements (inherited and somatic) were identified. The inherited aberrations in chromosome arms B, D, F as well as all somatic rearrangements (heterozygous inversions, deficiencies and deletions) were observed for the first time in this species. Asynapsis affected all chromosomes and a new region of activity in chromosome G, designated ‘Balbiani ring (BR2)’, was detected. Variations in the activity of the Balbiani rings and NOR were evaluated. Changes in the degree of ‘puffing’ in the telomere region of chromosome G are considered as a compensatory mechanism in response to the environmental conditions in the Afon Goch. As somatic chromosome rearrangements can be caused by stress agents we suggest that the somatic alterations in C. acidophilus indicate the existence of pollution-induced stress.

The mutation of the Suppressor of Underreplication gene does not affect the replication fork rate in the Drosophila melanogaster salivary gland polytene chromosomes

The mutation of the Suppressor of Underreplication gene does not affect the replication fork rate in the Drosophila melanogaster salivary gland polytene chromosomes

Functional analysis of Drosophila polytene chromosomes decompacted unit: the interband

Differential compaction of the interphase chromosomes is important for proper functioning of the eukaryotic genome. Such non-uniform compaction is most easily observed in Drosophila salivary gland polytene chromosomes as a reproducible banding pattern. Functional mechanisms underlying the establishment and maintenance of the banding pattern remain unclear but have been hypothesized to involve transcription and chromatin insulators. We tested functional properties of DNA fragments from several transcriptionally inert interband regions that behave as autonomous decompacted units of polytene chromosomes. Our results suggest that, in the absence of transcription, the decondensed state of interband regions does not depend on the presence of insulator elements but instead correlates with the presence of transcriptional enhancers.

Drosophila ISWI Regulates the Association of Histone H1 With Interphase Chromosomesin Vivo

Although tremendous progress has been made toward identifying factors that regulate nucleosome structure and positioning, the mechanisms that regulate higher-order chromatin structure remain poorly understood. Recent studies suggest that the ISWI chromatin-remodeling factor plays a key role in this process by promoting the assembly of chromatin containing histone H1. To test this hypothesis, we investigated the function of H1 in Drosophila. The association of H1 with salivary gland polytene chromosomes is regulated by a dynamic, ATP-dependent process. Reducing cellular ATP levels triggers the dissociation of H1 from polytene chromosomes and causes chromosome defects similar to those resulting from the loss of ISWI function. H1 knockdown causes even more severe defects in chromosome structure and a reduction in nucleosome repeat length, presumably due to the failure to incorporate H1 during replication-dependent chromatin assembly. Our findings suggest that ISWI regulates higher-order chromatin structure by modulating the interaction of H1 with interphase chromosomes.

GAL4 induces transcriptionally active puff in the absence of dSAGA- and ATAC-specific chromatin acetylation in the Drosophila melanogaster polytene chromosome

Puffs in the polytene chromosome of Drosophila melanogaster are characteristic of sites of high-level active transcription which can be observed directly under the microscope. We studied the dependence of puff formation on chromatin modifications at a site where a GAL4-inducible transgene is located in the 61C7 cytological region. Immunostaining of salivary gland polytene chromosomes indicated no increase of either dSAGA-specific histone H3 lysine 14, or ATAC-specific histone H4 lysine 5 and 12 acetylation in the puffed region. Nor did we observe increased levels of H4K8ac, H3K18ac, or H4K16ac in the puff. In accordance with the above, puff formation as well as localization of Pol II and GAL4 was detectable at the 61C region in dAda2b and dAda2a null homozygotes, which are dSAGA- and ATAC-specific mutants, respectively. Moreover, the reduced level of JIL-1-specific H3 serine 10 phosphorylation did not abolish puff formation in ATAC mutants. Surprisingly, in wild-type animals dADA3 and GCN5 shared constituents of dSAGA and ATAC, as well as JIL-1 localized specifically to the puff, where the JIL-1-phosphorylated H3S10ph level was also high. Altogether these data strongly suggest that the GAL4 activator can induce transcription and chromatin reorganization seen as a puff without dSAGA- and ATAC-specific histone acetylation and JIL-1-specific histone H3 phosphorylation.

Chironomus blaylocki sp. n. and C. bifurcatus sp. n., North American species near the base of the decorus-group (Diptera: Chironomidae)

Two species of the cytologically defined Chironomus decorus-group, C. bifurcatus sp. n. and C. blaylocki sp. n., are described on the basis of their salivary gland polytene chromosomes and larval morphology, with the associated male and pupa of C. bifurcatus and the putative male of C. blaylocki included as paratypes. The banding patterns of the salivary gland chromosomes indicate that these species are near the base of the cytologically defined decorus-group. The cytology and adults of these new species are compared with those of a number of other undescribed North American decorus-group species to demonstrate that they are distinct species.

Linker histone H1 is essential for Drosophila development, the establishment of pericentric heterochromatin, and a normal polytene chromosome structure.

We generated mutant alleles of Drosophila melanogaster in which expression of the linker histone H1 can be down-regulated over a wide range by RNAi. When the H1 protein level is reduced to approximately 20% of the level in wild-type larvae, lethality occurs in the late larval - pupal stages of development. Here we show that H1 has an important function in gene regulation within or near heterochromatin. It is a strong dominant suppressor of position effect variegation (PEV). Similar to other suppressors of PEV, H1 is simultaneously involved in both the repression of euchromatic genes brought to the vicinity of pericentric heterochromatin and the activation of heterochromatic genes that depend on their pericentric localization for maximal transcriptional activity. Studies of H1-depleted salivary gland polytene chromosomes show that H1 participates in several fundamental aspects of chromosome structure and function. First, H1 is required for heterochromatin structural integrity and the deposition or maintenance of major pericentric heterochromatin-associated histone marks, including H3K9Me(2) and H4K20Me(2). Second, H1 also plays an unexpected role in the alignment of endoreplicated sister chromatids. Finally, H1 is essential for organization of pericentric regions of all polytene chromosomes into a single chromocenter. Thus, linker histone H1 is essential in Drosophila and plays a fundamental role in the architecture and activity of chromosomes in vivo.

The heat shock 70 genes of the olive pest Bactrocera oleae: genomic organization and molecular characterization of a transcription unit and its proximal promoter region

A Bactrocera oleae genomic library was constructed and several genomic clones bearing hsp70 sequences were isolated. All clones were in situ hybridized on the major heat shock puff locus of the salivary gland polytene chromosomes. Restriction mapping of the isolated clones and genomic Southern hybridization indicated the presence of several putative hsp70 genes organized in a single cluster. Sequence analysis of an hsp70 transcription unit revealed a single 1905 nt long open reading frame that exhibits characteristic features of the inducible members of the HSP70 family. The presence and organization of many typical binding sites for the Heat Shock and GAGA factors suggest that the promoter of this gene is highly heat-inducible and could be used for conditional expression in transformation systems.

Misregulated RNA Pol II C-terminal domain phosphorylation results in apoptosis.

Misregulation of the level of RNA polymerase II carboxyl-terminal domain (CTD) phosphatase, Fcp1, in Drosophila results in high level of caspase-mediated apoptosis. Apoptosis induction by Fcp1 misregulation requires the presence of Drosophila melanogaster (Dm)p53, but occurs without the transcriptional activation of Dmp53 proapoptotic targets rpr, ark, and hid. Overproduction of a transcription activation-defective mutant Dmp53 protein increases, while Dmp53 null background decreases significantly the level of apoptosis in Fcp1-misregulated animals. Generating the apoptotic signal does not require the function of the ATM and Rad3-related kinase (ATR), and no significant level of nucleo-cytoplasmic translocation of Dmp53 is detectable in cells expressing Fcp1 at an abnormal level. Immunostaining of larval salivary gland polytene chromosomes with anti-Dmp53 antibodies indicates Dmp53 localization at several transcriptionally active chromosomal regions in wild-type cells, while in Fcp-misregulated cells the association of Dmp53 with specific chromosomal sites is decreased.

Karyotypes of Rare and Little-Known Species of Blackflies (Diptera, Simuliidae)

Cytomaps of polytene chromosomes of salivary glands of three species of Simuliidae, Cnetha Shutovae Rubzov, 1956 (2n = 6, 2n = 6+B), Eusimulium kazahstanicum Rubzov, 1976 (2n = 4) and Odagmia gribae Rubzov, 1956 (2n = 6) are presented. Karyological characteristics are given for each species. Yankovsky’s (2002) system of the family Simuliidae is used.

Umbrea, a chromo shadow domain protein in Drosophila melanogaster heterochromatin, interacts with Hip, HP1 and HOAP

Drosophila melanogaster HP1-interacting protein (Hip) is a partner of heterochromatin protein 1 (HP1) and is involved in transcriptional epigenetic gene silencing and the formation of heterochromatin. Recently, it has been shown that HP1 interacts with the telomere capping factor HP1/ORC (origin recognition complex)-associated protein (HOAP). Telomeres, complexes of DNA and proteins at the end of linear chromosomes, have been recognized to protect chromosome ends from degradation and fusion events. Both proteins are located at telomeres and prevent telomere fusions. Here, we report the identification and characterization of the Hip-interacting protein Umbrea. We found that Umbrea interacts directly with Hip, HP1 and HOAP in vitro. Umbrea, Hip and HP1 are partners in a protein complex in vivo and completely co-localize in the pericentric heterochromatin and at telomeres. Using a Gal4-induced RNA interference system, we found that after depletion of Umbrea in salivary gland polytene chromosomes, they exhibit multiple telomeric fusions. Taken together, these results suggest that Umbrea cooperates with Hip, HP1 and HOAP and plays a functional role in mediating normal telomere behaviour in Drosophila.

Mitotic and polytene chromosome analysis in the Mexican fruit fly,Anastrepha ludens(Loew) (Diptera: Tephritidae)

The present study constitutes the first attempt to construct a polytene chromosome map of an Anastrepha species, Anastrepha ludens (Loew), a major agricultural pest. The mitotic karyotype has a diploid complement of 12 acrocentric chromosomes, including five pairs of autosomes and an XX/XY sex chromosome pair. The analysis of salivary gland polytene chromosomes has shown a total number of five polytene elements that correspond to the five autosomes. The characteristic features and the most prominent landmarks of each chromosome are described. By comparing chromosome banding patterns, the possible chromosomal homology between A. ludens and Ceratitis capitata (Wiedemann) is presented. This work shows that polytene maps of A. ludens are suitable for cytogenetic studies in this species and may be used as reference for other Anastrepha species, most of which are also serious agricultural pests.

Genetic and cytogenetic analysis of the fruit flyRhagoletis cerasi(Diptera: Tephritidae)

The European cherry fruit fly, Rhagoletis cerasi, is a major agricultural pest for which biological, genetic, and cytogenetic information is limited. We report here a cytogenetic analysis of 4 natural Greek populations of R. cerasi, all of them infected with the endosymbiotic bacterium Wolbachia pipientis. The mitotic karyotype and detailed photographic maps of the salivary gland polytene chromosomes of this pest species are presented here. The mitotic metaphase complement consists of 6 pairs of chromosomes, including one pair of heteromorphic sex chromosomes, with the male being the heterogametic sex. The analysis of the salivary gland polytene complement has shown a total of 5 long chromosomes (10 polytene arms) that correspond to the 5 autosomes of the mitotic nuclei and a heterochromatic mass corresponding to the sex chromosomes. The most prominent landmarks of each polytene chromosome, the "weak points", and the unusual asynapsis of homologous pairs of polytene chromosomes at certain regions of the polytene elements are also presented and discussed.

The Chromatin Remodelling Factor dATRX Is Involved in Heterochromatin Formation

Despite extensive study of heterochromatin, relatively little is known about the mechanisms by which such a structure forms. We show that the Drosophila homologue of the human α-thalassemia and mental retardation X-linked protein (dATRX), is important in the formation or maintenance of heterochromatin through modification of position effect variegation. We further show that there are two isoforms of the dATRX protein, the longer of which interacts directly with heterochromatin protein 1 (dHP-1) through a CxVxL motif both in vitro and in vivo. These two proteins co-localise at heterochromatin in a manner dependent on this motif. Consistent with this observation, the long isoform of the dATRX protein localises primarily to the heterochromatin at the chromocentre on salivary gland polytene chromosomes, whereas the short isoform binds to many sites along the chromosome arms. We suggest that the establishment of a regular nucleosomal organisation may be common to heterochromatin and transcriptionally repressed chromatin in other locations, and may require the action of ATP dependent chromatin remodelling factors.

The Transcriptional Coactivator SAYP Is a Trithorax Group Signature Subunit of the PBAP Chromatin Remodeling Complex

SWI/SNF ATP-dependent chromatin remodeling complexes (remodelers) perform critical functions in eukaryotic gene expression control. BAP and PBAP are the fly representatives of the two evolutionarily conserved major subclasses of SWI/SNF remodelers. Both complexes share seven core subunits, including the Brahma ATPase, but differ in a few signature subunits; POLYBROMO and BAP170 specify PBAP, whereas OSA defines BAP. Here, we show that the transcriptional coactivator and PHD finger protein SAYP is a novel PBAP subunit. Biochemical analysis established that SAYP is tightly associated with PBAP but absent from BAP. SAYP, POLYBROMO, and BAP170 display an intimately overlapping distribution on larval salivary gland polytene chromosomes. Genome-wide expression analysis revealed that SAYP is critical for PBAP-dependent transcription. SAYP is required for normal development and interacts genetically with core- and PBAP-selective subunits. Genetic analysis suggested that, like BAP, PBAP also counteracts Polycomb silencing. SAYP appears to be a key architectural component required for the integrity and association of the PBAP-specific module. We conclude that SAYP is a signature subunit that plays a major role in the functional specificity of the PBAP holoenzyme.

Cell-Type-Specific TEV Protease Cleavage Reveals Cohesin Functions in Drosophila Neurons

SummaryCohesin is a highly conserved multisubunit complex that holds sister chromatids together in mitotic cells. At the metaphase to anaphase transition, proteolytic cleavage of the α kleisin subunit (Rad21) by separase causes cohesin's dissociation from chromosomes and triggers sister-chromatid disjunction. To investigate cohesin's function in postmitotic cells, where it is widely expressed, we have created fruit flies whose Rad21 can be cleaved by TEV protease. Cleavage causes precocious separation of sister chromatids and massive chromosome missegregation in proliferating cells, but not disaggregation of polytene chromosomes in salivary glands. Crucially, cleavage in postmitotic neurons is lethal. In mushroom-body neurons, it causes defects in axon pruning, whereas in cholinergic neurons it causes highly abnormal larval locomotion. These data demonstrate essential roles for cohesin in nondividing cells and also introduce a powerful tool by which to investigate protein function in metazoa.

The Drosophila jumonji gene encodes a JmjC-containing nuclear protein that is required for metamorphosis

Jumonji (Jmj) is a transcriptional repressor that plays important roles in the suppression of cell proliferation and development of various tissues in the mouse. To further clarify the roles of Jmj during development and gain insight into mechanisms of Jmj-mediated transcriptional regulation, we have taken advantage of Drosophila as a model organism. Drosophila Jmj (dJmj) shares high homology with mammalian Jmj in the JmjN, JmjC and AT-rich interaction domains, as well as in the N-terminal repression domain. dJmj localizes to hundreds of euchromatic sites but not to chromocenter heterochromatin on salivary gland polytene chromosomes. In addition, dJmj is excluded from regions stained with an antibody against Ser5-phosphorylated RNA polymerase II, suggesting a function of dJmj in transcriptionally inactive chromatin. Loss of djmj results in larval and pupal lethality with phenotypes similar to those observed in mutants of ecdysone-regulated genes, implying the involvement of dJmj in the repression of gene expression in the ecdysone pathway. Transgenic mouse Jmj mostly colocalizes with dJmj and partially rescues the phenotypes of djmj mutants, indicating that dJmj is a functional homolog of mammalian Jmj. Furthermore, mutation in djmj suppresses position effect variegation of the T(2;3)Sb(V) rearrangement. These findings suggest that dJmj controls expression of developmentally important genes through modification of chromatin into a transcriptionally silenced state.

An integrated genetic and cytogenetic map for the Mediterranean fruit fly, Ceratitis capitata, based on microsatellite and morphological markers

A genetic map based on microsatellite polymorphisms and visible mutations of the Mediterranean fruit fly (medfly), Ceratitis capitata is presented. Genotyping was performed on single flies from several backcross families. The map is composed of 67 microsatellites and 16 visible markers distributed over four linkage groups. Fluorescence in situ hybridization of selected microsatellite markers on salivary gland polytene chromosomes allowed the alignment of these groups to the second, fourth, fifth and sixth chromosome. None of the markers tested showed segregation either with the X or the third chromosome. However, this map constitutes a substantial starting point for a detailed genetic map of C. capitata. The construction of an integrated map covering the whole genome should greatly facilitate genetic studies and future genome sequence projects of the species.

High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. Here, we describe how, in double mutants for Su(var)3-9 and SuUR genes encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed.

Interbands behave as decompacted autonomous units in Drosophila melanogaster polytene chromosomes

We studied whether interbands can be ectopically formed in Drosophila melanogaster polytene chromosomes. For comparative purposes, two types of P-element constructs were used. The first type was represented by P-element based insertions into compact bands. Sequences of these insertions or adjacent genomic sequences could be activated ectopically either by GAL4 or by dosage compensation machinery. In the second type, the DNA from transcriptionally silent interbands was positioned between the FRT sites, and was flanked by DNA sequences of genes that were also inactive in salivary glands. Electron microscopy analysis of salivary gland polytene chromosomes demonstrated that both types of constructs formed distinct, yet morphologically similar interbands. Notably, the second class of transposon insertions appeared in polytene chromosomes as two bands separated by one interband. Excision of interband material from such insertions resulted in fusion of newly appeared bands into a single band. We were able to confirm by molecular means that the DNA sequences in integrated constructs were intact, that chromatin organization of this DNA mimicked that of native interbands, and that it was accurately excised from the constructs by FLP. Thus, we demonstrate that transfer of interband DNA into a silent genetic environment does not compromise interband formation. Our results do not support the idea of the existence of distinct cytogenetic "band + interband" units, furthermore, they suggest the autonomy of the decompacted state of interbands.