Regions Of Polytene Chromosomes Research Articles

Influence of the SuUR gene on intercalary heterochromatin in Drosophila melanogaster polytene chromosomes.

Salivary gland polytene chromosomes of Drosophila melanogaster have a reproducible set of intercalary heterochromatin (IH) sites, characterized by late DNA replication, underreplicated DNA, breaks and frequent ectopic contacts. The SuUR mutation has been shown to suppress underreplication, and wild-type SuUR protein is found at late-replicating IH sites and in pericentric heterochromatin. Here we show that the SuUR gene influences all four IH features. The SuUR mutation leads to earlier completion of DNA replication. Using transgenic strains with two, four or six additional SuUR(+) doses (4-8xSuUR(+)) we show that wild-type SuUR is an enhancer of DNA underreplication, causing many late-replicating sites to become underreplicated. We map the underreplication sites and show that their number increases from 58 in normal strains (2xSuUR(+)) to 161 in 4-8xSuUR(+) strains. In one of these new sites (1AB) DNA polytenization decreases from 100% in the wild type to 51%-85% in the 4xSuUR (+) strain. In the 4xSuUR(+) strain, 60% of the weak points coincide with the localization of Polycomb group (PcG) proteins. At the IH region 89E1-4 (the Bithorax complex), a typical underreplication site, the degree of underreplication increases with four doses of SuUR(+) but the extent of the underreplicated region is the same as in wild type and corresponds to the region containing PcG binding sites. We conclude that the polytene chromosome regions known as IH are binding sites for SuUR protein and in many cases PcG silencing proteins. We propose that these stable silenced regions are late replicated and, in the presence of SuUR protein, become underreplicated.

Genetic analysis of Drosophila melanogaster polytene chromosome region 44D-45F: loci required for viability and fertility.

A genetic screen to identify mutations in genes in the 45A region on the right arm of chromosome 2 that are involved in oogenesis in Drosophila was undertaken. Several lethal but no female sterile mutations in the region had previously been identified in screens for P-element insertion or utilizing X rays or EMS as a mutagen. Here we report the identification of EMS-induced mutations in 21 essential loci in the 45D-45F region, including 13 previously unidentified loci. In addition, we isolated three mutant alleles of a newly identified locus required for fertility, sine prole. Mutations in sine prole disrupt spermatogenesis at or before individualization of spermatozoa and cause multiple defects in oogenesis, including inappropriate division of the germline cyst and arrest of oogenesis at stage 4.

The Drosophila Suppressor of Underreplication Protein Binds to Late-Replicating Regions of Polytene Chromosomes

In many late-replicating euchromatic regions of salivary gland polytene chromosomes, DNA is underrepresented. A mutation in the SuUR gene suppresses underreplication and leads to normal levels of DNA polytenization in these regions. We identified the SuUR gene and determined its structure. In the SuUR mutant stock a 6-kb insertion was found in the fourth exon of the gene. A single SuUR transcript is present at all stages of Drosophila development and is most abundant in adult females and embryos. The SuUR gene encodes a protein of 962 amino acids whose putative sequence is similar to the N-terminal part of SNF2/SWI2 proteins. Staining of salivary gland polytene chromosomes with antibodies directed against the SuUR protein shows that the protein is localized mainly in late-replicating regions and in regions of intercalary and pericentric heterochromatin.

The JIL-1 Tandem Kinase Mediates Histone H3 Phosphorylation and Is Required for Maintenance of Chromatin Structure in Drosophila

To analyze the function of the chromosomal kinase JIL-1, we generated an allelic series of hypomorphic and null mutations. JIL-1 is an essential kinase for viability, and reduced levels of JIL-1 kinase activity lead to a global change in chromatin structure. In JIL-1 hypomorphs, euchromatic regions of polytene chromosomes are severely reduced and the chromosome arms condensed. This is correlated with decreased levels of histone H3 Ser10 phosphorylation. These levels can be restored by a JIL-1 transgene placing JIL-1 directly in the pathway mediating histone H3 phosphorylation. We propose a model where JIL-1 kinase activity is required for maintaining the structure of the more open chromatin regions that facilitate gene transcription.

JIL-1: A Novel Chromosomal Tandem Kinase Implicated in Transcriptional Regulation in Drosophila

We have cloned and characterized JIL-1, a novel tandem kinase in Drosophila that associates with the chromosomes throughout the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost 2-fold on the hypertranscribed male X chromosome compared to autosomes. Phylogenetic analysis suggests that JIL-1 together with human MSKs defines a separate family of tandem kinases. That JIL-1 is a functional kinase was demonstrated by autophosphorylation and phosphorylation of histone H3 in vitro. Based on these findings, we propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure.

Interbands ofDrosophila melanogaster polytene chromosomes contain matrix association regions

The DNA of three previously cloned interband regions (85D9/D10, 86B4/B6, and 61C7/C8) of Drosophila melanogaster polytene chromosomes has been tested for the presence of matrix association regions (MAR), using the in vitro matrix-binding assay of Cockerill and Garrard. MARs were found in all three interband regions under study. These results are discussed in frames of a model postulating that interband regions of polytene chromosomes correspond to the chromosomal DNA loop borders, which can be identified in interphase nuclei using biochemical approaches.

Chromatin structure and nuclear remodeling.

Nuclear architecture is remodeled during interphase in response to changes in gene activity as well as to changing structural and functional requirements during cell division. Using the monoclonal antibody mAb2A, we have identified two proteins that appear to play important roles in these processes: JIL-1 is a tandem serine-threonine kinase implicated in the regulation of chromatin structure, whereas Skeletor is a novel protein participating in structural nuclear remodeling during the cell cycle. Antibody staining and live imaging of JIL-1-GFP transgenic flies show that JIL-1 localizes to the gene-rich interband regions of larval polytene chromosomes and is upregulated almost twofold on the hypertranscribed male X chromosome compared with autosomes. We propose that JIL-1 may play a role in transcriptional control potentially by regulating chromatin structure. The other mAb2A antigen, Skeletor, is distributed in a nuclear meshwork pattern that can be observed in stereo pair images to reorganize during the cell cycle to form a spindle-like structure at prometaphase that is distinct from the microtubule spindle apparatus. Taking advantage of the powerful molecular and genetic approaches offered in Drosophila, the study of these two proteins promises to yield new insight into what defines nuclear architecture at the molecular level and how its remodeling is regulated.

Critical electrolyte concentration of chromatin in polytene chromosomes of Trichosia pubescens (Diptera, Sciaridae)

The binding of toluidine blue molecules to nucleic acid phosphates under conditions of competition with Mg2+ ions was studied in Trichosia pubescens polytene chromosome regions differing in composition and organization (RNA-rich areas and puffs, DNA puffs, heterochromatin). The aim was to find variability in the Mg2+ concentration at which metachromasy was completely prevented (= critical electrolyte concentration; CEC) and that could reflect differences at the level of nucleoprotein complexes in particular chromosome regions and developmental phases. Although high CEC values have been found in tightly packed chromatin such as that of heterochromatin zones, the CEC values for the other regions have proven to be affected not only by availability and proximity of DNA phosphates but also by RNA richness and other factors. Among these, changes in DNA geometry and packing state have been assumed for explaining increased values after RNA removal in DNA puffs and RNA-rich zones. Based on CEC values it has been suggested that alterations at the level of nucleoprotein complexes may occur in puffs before they are morphologically detectable. CEC results for polytene chromosomes were thus revealed to vary considerably with slight variations in nucleoprotein composition and organization. However, since a complex and apparently contradictory rationale has to be used for explaining part of the results, CEC is not recommended as a useful tool for extensive and comparative studies of this particular model.

Ultrastructure of regions containing homologous loci in polytene chromosomes of Drosophila melanogaster and Drosophila subobscura.

We have used a new approach involving in situ hybridisation and electron microscopy to establish ultrastructural homologies between polytene chromosome regions of Drosophila melanogaster and Drosophila subobscura. Twelve probes were chosen to cover all the chromosomal elements: the myospheroid gene, the collagen type IV gene, the collagen-like gene, the w26 homeobox gene, the beta3 tubulin gene, the kinesin heavy chain gene, the tryptophan hydrolase gene, the Hsp82, Hsp22-26 and Hsp23-28, Hsp68, Hsp70 genes and the beta unit of the F0-F1 ATPase gene. Most of these loci were previously undescribed in D. subobscura and imprecisely located in D. melanogaster. We have demonstrated here, by an ultrastructural analysis of each chromosomal region, that homologous genetic loci tend to show a similar ultrastructure in the two species. With a few exceptions, the structural homology extends to the chromosomal regions surrounding the loci. In some cases, however, no structurally recognisable homology can be seen either in the locus or in its flanking regions.

A 1.5 kb repeat sequence flanks the suppressor of forked gene at the euchromatin-heterochromatin boundary of the Drosophila melanogaster X chromosome.

A 1.5 kilobasepair repeated DNA sequence is duplicated in direct orientation so as to flank the suppressor of forked gene in the euchromatin-heterochromatin transition region on the X chromosome of Drosophila melanogaster. These two copies are almost identical, but DNA blotting, analysis of cloned sequences and database searches show that elsewhere in the genome, homologous sequences are poorly conserved. They are often associated with other repeats, suggesting that they may belong to a scrambled and clustered middle repetitive DNA family. The sequences do not appear to be related to transposable elements and their location in different strains is conserved. In situ hybridization to metaphase chromosomes shows that homologous sequences are concentrated in the pericentric regions of the autosomes and the X chromosome. The sequences are not significantly under-represented in DNA from polytene tissue and must lie in the replicated regions of polytene chromosomes. The almost perfect conservation of the two repeats around suppressor of forked in D. melanogaster suggests they arose by duplication or gene conversion. Suppression of recombination in this chromosomal region presumably allows this unusual organization to be stably maintained. In the X-ray induced allele, suppressor of forked-L26, the sequence between the repeats, including the gene, and one copy of the repeat have been deleted.

Genetic analysis of oxygen defense mechanisms in Drosophila melanogaster and identification of a novel behavioural mutant with a Shaker phenotype.

Mutants of Drosophila melanogaster that lack Cu/Zn superoxide dismutase or urate are hypersensitive to reactive oxygen species (ROS) generated in vivo by the redox-cycling agent paraquat. We have subsequently employed paraquat as a selective agent to identify adult viable mutants potentially defective in other, perhaps unknown, components of ROS metabolism. Paraquat screening of ethyl methanesulfonate-induced second- and third-chromosome mutations yielded 24 paraquat hypersensitive mutants. Two mutants were identified as being new alleles of the previously identified doublesex (dsx) and pink (p) genes. The remainder of the mutations identified previously undescribed genes, including one second chromosome paraquat hypersensitive mutant that was found to exhibit shaking legs, abdomen pulsations, and body shuddering under ether anaesthesia. This recessive mutation was mapped to the polytene chromosome region of 48A5-48B2 and defines a new gene we named quiver (qvr). This mutation is similar in phenotype to the Shaker (Sh), ether-a-gogo (eag), and Hyperkinetic (Hk) mutations, all of which affect potassium channel function in D. melanogaster.

Immunofluorescent localization of nuclear protein in polytene chromosomes of two chironomid species:Endochironomus tendens(Fabr.) andGlyptotendipes gripekoveniKieffer (Diptera, Chironomidae)

SUMMARYA monoclonal antibody (Mab 2a8) has been generated against a DNA tight binding protein of eukaryotic cells. Its distribution was investigated in salivary gland polytene chromosomes of two Chironomid species, Endochironomus tendens and Glyptotendipes gripekoveni, by immunofluorescence. The results indicate: 1. Mab 2a8 is distributed in some distinct condensed bands along the entire lenght of chromosomes; 2. The banding pattern obtained with fluorescent antibody does almost strictly correspond to some Hoechst positive bands and to constitutive heterochromatin sites established by «C» banding method. Other condensed (Hoechst positive) bands on chromosomes were Mab 2a8 immunonegative. 3. No immunofluorescent was observed in genetically active regions of polytene chromosomes (puffs, Balbiani rings and nuclear organizer). The mode of distribution in defined bands of polytene chromosomes suggests the residence of this protein in polytene chromosomes, whose presence appears to be well in correlation with t...

The faint band/interband region 28C2 to 28C4-5(−) of the Drosophila melanogaster salivary gland polytene chromosomes is rich in transcripts

Urate oxidase mRNA and five other transcripts map along 38 kb of DNA in the region 28C on the Drosophila melanogaster second chromosome. Three biotinylated restriction fragments from this 38 kb of DNA, one from each end and one from the middle, were individually hybridized in situ to slightly stretched salivary gland polytene chromosomes. The data from these in situ hybridizations in combination with the transcription map of the 38 kb of DNA indicate that: (i) there are six discrete RNA species encoded along the 38 kb of DNA and (ii) these six transcripts map to the faint band/interband region which includes the proximal edge of 28C1, the three faint bands, 28C2, 28C3 and 28C4-5(-), and the adjacent interband chromatin. Our data are consistent with the few published studies directly demonstrating that faint band/interband regions of the Drosophila melanogaster salivary gland polytene chromosomes code for a high density of transcripts.

Genetic analysis of the adenosine3 (Gart) region of the second chromosome of Drosophila melanogaster.

The Gart gene of Drosophila melanogaster is known, from molecular biological evidence, to encode a polypeptide that serves three enzymatic functions in purine biosynthesis. It is located in polytene chromosome region 27D. One mutation in the gene (ade3(1)) has been described previously. We report here forty new ethyl methanesulfonate-induced mutations selected aga!nst a synthetic deficiency of the region from 27C2-9 to ++28B3-4. The mutations were characterized cytogenetically and by complementation analysis. The analysis apparently identifies 12 simple complementation groups. In addition, two segments of the chromosome exhibit complex complementation behavior. The first, the 28A region, gave three recessive lethals and also contains three known visible mutants, spade (spd), Sternopleural (Sp) and wingless (wg); a complex pattern of genetic interaction in the region incorporates both the new and the previously known mutants. The second region is at 27D, where seven extreme semilethal mutations give a complex complementation pattern that also incorporates ade3(1). Since ade3(1) is defective in one of the enzymatic functions encoded in the Gart gene, we assume the other seven also affect the gene. The complexity of the complementation pattern presumably reflects the functional complexity of the gene product. The phenotypic effects of the mutants at 27D are very similar to those described for ade2 mutations, which also interrupt purine biosynthesis.

Electron microscopical analysis of Drosophila polytene chromosomes

An electron microscopical (EM) analysis was performed on regions of polytene chromosomes which contained DNA segments of different genetic composition, inserted by P element-mediated transformation into the Drosophila melanogaster genome. In seven of ten regions examined, containing insertions of the hsp28-ry, hsp70-Adh, ryhsp 70-beta-gal genes and of the ry gene tetramer, new bands appeared. Lack of new bands in three other strains is apparently connected with the fusion of the inserted material to preexisting bands. The new bands do not differ morphologically from the usual bands of polytene chromosomes, and their formation is likely due to predominant insertion of DNA segments into interbands. Among the constructs examined, the minimal length of a DNA segment which appears as a new band is about 5 kb; the DNA packing ratio in the new bands varies from 30 to 50. Activation of the inserted genes by heat shock has enabled us to observe the puffing characteristics of new bands. A sequence of some one kb forms a large interband, or micropuff; the puff size is correlated with the length of the genes being activated. If a DNA segment contains a single gene, then its activation causes the decompaction of the whole band; however, when a DNA segment consists of two genes and the promoter element of the activated gene is positioned in the middle of the sequence, the band splits and only part is decompacted and puffed. The DNA packing ratio in the puffs is 1.4-3.5. The subsequent deletion of the hsp70 promoter but retention of 23, 59, and 73 by from the transcription start points leads to failure of puff formation. In all the transformed sites an increase in the total length of the interbands adjacent to the insert as compared with the initial interband was observed. This increase appears to be due to decompaction of the P element DAN flanking the inserted segments. It is shown that a DNA segment, consisting of four tandemly repeated ry gene copies and interspersed by material which includes P DNA, forms a complex of loose chromatin in which, however, four bands can be resolved. We also observed a lengthening of interband regions containing only the P element sequence itself. Insertion of the complete 2.9 kb P element into the large single 10A1-2 bound of the X chromosome (an insertion in the region approximately 10 kb to the right of the v gene) causes splitting of the band into two parts and formation of a new interband.(ABSTRACT TRUNCATED AT 400 WORDS)

Levels of RNA from a Family of Putative Serine Protease Genes Are Reduced in Drosophila melanogaster dunce Mutants and Are Regulated by Cyclic AMP

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.

Levels of RNA from a family of putative serine protease genes are reduced in Drosophila melanogaster dunce mutants and are regulated by cyclic AMP.

We have isolated several genes expressed at abnormal levels in the memory mutant, dunce (dnc), of Drosophila melanogaster. These mutants have an elevated cyclic AMP (cAMP) content due to a mutation in the structural gene for cAMP phosphodiesterase, so the isolated genes are potentially ones regulated by cAMP. Here, we describe the characterization of a genomic clone and corresponding cDNA clones which contain sequences that are underexpressed in dnc mutants. Sequence analysis of portions of the genomic clone and representative cDNAs revealed the presence of two uninterrupted and complete open reading frames (SER1 and SER2) and part of a third (SER3). The predicted amino acid sequences of all of these were found to be homologous to the serine protease family of enzymes. The genomic clone was localized to the polytene chromosome region 99C-D, although genome-blotting experiments indicated the existence of several other genes related to the cloned serine protease-like genes. Hybridization experiments with probes representing each of the three sequenced genes showed that only the SER1-related genes were differentially expressed in dnc mutants. The putative serine protease genes were abundantly expressed in the larval gut, suggesting a major function in digestion. Feeding normal flies cAMP, isobutylmethylxanthine, or forskolin resulted in a decreased RNA level of the SER1-related genes. Thus, RNA levels of this serine protease gene family are negatively regulated by cAMP.

P75, a polypeptide component of karyoskeletal protein-enriched fractions associated with transcriptionally active loci of Drosophila melanogaster polytene chromosomes.

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

P75, a Polypeptide Component of Karyoskeletal Protein-Enriched Fractions Associated with Transcriptionally Active Loci of Drosophila melanogaster Polytene Chromosomes

A 75-kilodalton polypeptide has been identified which copurifies with karyoskeletal protein-enriched fractions prepared from Drosophila melanogaster embryos. Results of indirect immunofluorescence experiments suggest that this protein, here designated p75, is primarily associated with puffed regions of larval salivary gland polytene chromosomes. In nonpolytenized Schneider 2 tissue culture cells, p75 appeared to be localized throughout the nuclear interior during interphase. In mitotic cells, p75 was redistributed diffusely. A possible role for karyoskeletal elements in transcriptional regulation is discussed.

Microcloning reveals a high frequency of repetitive sequences characteristic of chromosome 4 and the beta-heterochromatin of Drosophila melanogaster.

Microdissection and microcloning of the euchromatin-heterochromatin transition region of the Drosophila melanogaster polytene X chromosome and part of the euchromatin of chromosome 4 reveals that they share certain features characteristic of beta-heterochromatin, which is morphologically defined as the loosely textured material at the bases of some polytene chromosome arms. Both are mosaics of many different middle-repetitive DNA sequences interspersed with single-copy DNA sequences. Sixty percent of cloned inserts derived from division 20 and about 40 percent from subdivisions 19EF of the X chromosome harbor at least one repetitive DNA sequence in an average insert of 4.5 kilobases. No repeats have significant cross-hybridization to any of the eleven satellite DNAs, or to the clustered-scrambled sequences present in pDm1. The repetitive elements are, in general, confined to the beta-heterochromatic regions of polytene chromosomes, but some are adjacent to nomadic elements. Chromosome 4, however, has some repeats spread throughout its entire euchromatin. These data have implications for the structure of transition zones between euchromatin and heterochromatin of mitotic chromosomes and also provide a molecular basis for reexamining some of the unusual classical properties of chromosome 4.