Polytene Bands Research Articles

Biased visibility in Hi-C datasets marks dynamically regulated condensed and decondensed chromatin states genome-wide

BackgroundProximity ligation based techniques, like Hi-C, involve restriction digestion followed by ligation of formaldehyde cross-linked chromatin. Distinct chromatin states can impact the restriction digestion, and hence the visibility in the contact maps, of engaged loci. Yet, the extent and the potential impact of digestion bias remain obscure and under-appreciated in the literature.ResultsThrough analysis of 45 Hi-C datasets, lamina-associated domains (LADs), inactive X-chromosome in mammals, and polytene bands in fly, we first established that the DNA in condensed chromatin had lesser accessibility to restriction endonucleases used in Hi-C as compared to that in decondensed chromatin. The observed bias was independent of known systematic biases, was not appropriately corrected by existing computational methods, and needed an additional optimization step. We then repurposed this bias to identify novel condensed domains outside LADs, which were bordered by insulators and were dynamically associated with the polycomb mediated epigenetic and transcriptional states during development.ConclusionsOur observations suggest that the corrected one-dimensional read counts of existing Hi-C datasets can be reliably repurposed to study the gene-regulatory dynamics associated with chromatin condensation and decondensation, and that the existing Hi-C datasets should be interpreted with cautions.

Polytene Chromosome Structure and Somatic Genome Instability.

Polytene chromosomes have for 80 years provided the highest resolution view of interphase genome structure in an animal cell nucleus. These chromosomes represent the normal genomic state of nearly all Drosophila larval and many adult cells, and a better understanding of their striking banded structure has been sought for decades. A more recently appreciated characteristic of Drosophila polytene cells is somatic genome instability caused by unfinished replication (UR). Repair of stalled forks generates enough deletions in polytene salivary gland cells to alter 10%-90% of the DNA strands within more than 100 UR regions comprising 20% of the euchromatic genome. We accurately map UR regions and show that most approximate large polytene bands, indicating that replication forks frequently stall near band boundaries in late S phase. Chromosome conformation capture has recently identified dense topologically associated domains (TADs) in many genomes and most UR bands are similar or slightly smaller than a cognate Drosophila TAD. We argue that bands serve the evolutionarily ancient function of coordinating genome replication with local gene activity. We also discuss the relatively recent evolution of polyteny and somatic instability in Diptera and propose that these processes helped propel the amazing success of two-winged flies in becoming the most ecologically diverse insect group, with 200 times the number of species as mammals.

An integrated description of polytene chromosome structure

An integrated description from nucleohistone fiber level to light microscopic level of polytene chromosomes is presented. The integration is based on the results obtained by different new applications of electron microscopic techniques and on the analysis of earlier data of chromatin structure. A model for the organization of polytene chromosomes in vivo is extrapolated from the EM-data by approximating the effects of the physico-chemical parameters changing during preparative procedures. The nucleosome unit fiber forms loops, which are organized into about 500A diameter coils with 24 nucleosomes per turn of the double fiber, leading to an overall packing ratio of about 1:78. The coils are packed differentially into structural units forming the polytene bands, which are separated from each other by interbands of unit fibers with no higher order coiling evident. For the general higher order structure of chromatin a model capable of explaining former observations in other eukaryotic chromosome systems is proposed. A whole mount method suitable for nucleosome fiber examination in polytene chromosomes is introduced. A sectioning method preserving the nucleosomal structures and a quick method for whole mounting polytene chromosomes retaining their light microscopic morphology are also described.

Effects of mutations at the stambh A locus of Drosophila melanogaster.

We report novel findings on the cytogenetic location, functional complexity and maternal and germline roles of the stambh A locus of Drosophila melanogaster. stmA is localized to polytene bands 44D1.2 on 2R. stmA mutations are of two types: temperature-sensitive (ts) adult and larval paralytic or unconditional embryonic or larval lethal. Twelve alleles reported in this study fall into two intragenic complementing groups suggesting that stmA is a complex locus with more than one functional domain. Some unconditional embryonic lethal alleles show a 'neurogenic' phenotype of cuticle loss accompanied by neural hypertrophy. It is shown that embryos of ts paralytic alleles also show mild neural hypertrophy at permissive temperatures while short exposure to heat induces severe cuticle loss in these embryos. stmA exerts a maternal influence over heat-induced cuticle loss. Unconditional embryonic lethal alleles of stmA are also germline lethal.

Generation of a Semi-Dominant Mutation with Temperature Sensitive Effects on Both Locomotion and Phototransduction in Drosophila Melanogaster

A novel Drosophila mutant named Tripped-and-fell (Taf)) was isolated in a F1 screen for dominant temperature sensitive paralytics. Recombination mapping using multiply marked chromosomes and P elements have pinpointed the locus of Taf to polytene band 93 on the right arm of the third chromosome (3R). When exposed to restrictive temperatures, both Taf heterozygotes and homozygotes paralyzed; however, homozygotes paralyzed at lower temperatures and took longer to recover than heterozygotes. There are also positive correlations between recovery time from paralysis and both duration and temperature of exposure. Electroretinograms (ERGs) revealed that both homozygotes and heterozygotes have a grossly normal light response at 22°C, but at 37°C, the ERGs from both homozygotes and heterozygotes are unable to maintain a normal sustained depolarization and have ar educed off-transient potential. The severity of the ERG repolarization phenotype is greater in homozygotes than in heterozygotes.

Deletion of an insulator element by the mutation facet-strawberry in Drosophila melanogaster.

Eukaryotic chromosomes are thought to be subdivided into a series of structurally and functionally independent units. Critical to this hypothesis is the identification of insulator or boundary elements that delimit chromosomal domains. The properties of a Notch mutation, facet-strawberry (fa(swb)), suggest that this small deletion disrupts such a boundary element. fa(swb) is located in the interband separating polytene band 3C7, which contains Notch, from the distal band 3C6. The fa(swb) mutation alters the structural organization of the chromosome by deleting the interband and fusing 3C7 with 3C6. Genetic studies also suggest that fa(swb) compromises the functional autonomy of Notch by allowing the locus to become sensitive to chromosomal position effects emanating from distal sequences. In the studies reported here, we show that a DNA fragment spanning the fa(swb) region can insulate reporter transgenes against chromosomal position effects and can block enhancer-promoter interactions. Moreover, we find that insulating activity is dependent on sequences deleted in fa(swb). These results provide evidence that the element defined by the fa(swb) mutation corresponds to an insulator.

The characterization of DINE-1, a short, interspersed repetitive element present on chromosome and in the centric heterochromatin of Drosophila melanogaster.

The banded portion of chromosome 4 (the "dot" chromosome) in Drosophila melanogaster displays some properties of beta-heterochromatin, which is normally found within the centric domain of the chromosomes. The nature and distribution of repetitive elements on chromosome 4 could play a role in the establishment of this unusual chromatin configuration. We describe here one such element: a short, interspersed repetitive sequence named DINE-1. Determination of a consensus sequence for the element reveals that there are two conserved regions (A and B) separated by a highly variable spacer. The conserved sequences are approximately 400 bp long but degenerate at both ends, opening the possibility that a yet-to-be-discovered mother element may be present in the genome. DINE-1 bears few of the properties of the mammalian short interspersed elements (SINEs) to which it bears a superficial resemblance in size. It does not appear to be the product of reverse transcription and lacks any polymerase III promoter consensus. The elements are not flanked by target site duplications and their termini lack direct or inverted repeats, suggesting that they themselves are not transposable. Our analysis of cosmid clones from chromosome 4, and elsewhere in the genome, revealed that the euchromatic locations of DINE-1 are almost exclusively confined to chromosome 4. In situ hybridization of a DINE-1 probe to polytene chromosomes confirmed the preferential distribution along 4, in addition to its presence in the centric heterochromatin. This unusual genomic distribution of bias toward chromosome 4 is also seen in the sibling species, D. simulans, whose dot chromosomes exhibit poorly resolved polytene bands and lack crossing over during meiosis like those of D. melanogaster. However, the dot chromosome of D. virilis, which exhibits a well-defined banded structure on polytene chromosomes and can cross over, has only a single, discrete site of DINE-1 element hybridization. The presence of DINE-1 within these regions suggests a role in the heterochromatic nature of chromosome 4 in D. melanogaster and supports the contention that repeats accumulate in regions of diminished crossing over.

An exploration of the sequence of a 2.9-Mb region of the genome of Drosophila melanogaster: the Adh region.

A contiguous sequence of nearly 3 Mb from the genome of Drosophila melanogaster has been sequenced from a series of overlapping P1 and BAC clones. This region covers 69 chromosome polytene bands on chromosome arm 2L, including the genetically well-characterized "Adh region." A computational analysis of the sequence predicts 218 protein-coding genes, 11 tRNAs, and 17 transposable element sequences. At least 38 of the protein-coding genes are arranged in clusters of from 2 to 6 closely related genes, suggesting extensive tandem duplication. The gene density is one protein-coding gene every 13 kb; the transposable element density is one element every 171 kb. Of 73 genes in this region identified by genetic analysis, 49 have been located on the sequence; P-element insertions have been mapped to 43 genes. Ninety-five (44%) of the known and predicted genes match a Drosophila EST, and 144 (66%) have clear similarities to proteins in other organisms. Genes known to have mutant phenotypes are more likely to be represented in cDNA libraries, and far more likely to have products similar to proteins of other organisms, than are genes with no known mutant phenotype. Over 650 chromosome aberration breakpoints map to this chromosome region, and their nonrandom distribution on the genetic map reflects variation in gene spacing on the DNA. This is the first large-scale analysis of the genome of D. melanogaster at the sequence level. In addition to the direct results obtained, this analysis has allowed us to develop and test methods that will be needed to interpret the complete sequence of the genome of this species. Before beginning a Hunt, it is wise to ask someone what you are looking for before you begin looking for it. Milne 1926

Overexpression Beadex mutations and loss-of-function heldup-a mutations in Drosophila affect the 3' regulatory and coding components, respectively, of the Dlmo gene.

LIM domains function as bridging modules between different members of multiprotein complexes. We report the cloning of a LIM-containing gene from Drosophila, termed Dlmo, which is highly homologous to the vertebrate LIM-only (LMO) genes. The 3' untranslated (UTR) of Dlmo contains multiple motifs implicated in negative post-transcriptional regulation, including AT-rich elements and Brd-like boxes. Dlmo resides in polytene band 17C1-2, where Beadex (Bx) and heldup-a (hdp-a) mutations map. We demonstrate that Bx mutations disrupt the 3'UTR of Dlmo, and thereby abrogate the putative negative control elements. This results in overexpression of Dlmo, which causes the wing scalloping that is typical of Bx mutants. We show that the erect wing phenotype of hdp-a results from disruption of the coding region of Dlmo. This provides molecular grounds for the suppression of the Bx phenotype by hdp-a mutations. Finally, we demonstrate phenotypic interaction between the LMO gene Dlmo, the LIM homeodomain gene apterous, and the Chip gene, which encodes a homolog of the vertebrate LIM-interacting protein NLI/Ldb1. We propose that in analogy to their vertebrate counterparts, these proteins form a DNA-binding complex that regulates wing development.

The organization of the alpha-tubulin gene family in the Drosophila montium subgroup of the melanogaster species group.

The alpha 1-, alpha 2-, alpha 3-, and alpha 4-tubulin genes have been mapped by in situ hybridization to the polytene chromosomes of five species representative of the Drosophila montium subgroup geographical distribution. A lambda phage clone containing alpha 1-tubulin specific sequences was isolated from a genomic DNA library of Drosophila auraria and its restriction endonuclease pattern is presented. Both well-characterized heterologous and homologous probes were used to assess orthogonality of gene members between species groups. The in situ hybridization pattern observed in all species studied is consistent with that of Drosophila melanogaster, since alpha 1-, alpha 2-, and alpha 3-tubulin genes are located on the same polytene arm, and the alpha 4-tubulin gene is found on a different arm. Cross-hybridization was observed among alpha 1-, alpha 2-, and alpha 3-tubulin specific sequences in all species studied, using either heterologous or homologous probes. However, unlike D. melanogaster, in all montium species studied, both alpha 1- and alpha 3-tubulin specific probes hybridize to the same polytene band, indicating a clustered organization of the above genes. The chromosomal organization of this gene family would suggest that taxa within the montium subgroup are closer to their common ancestors than are the taxa in the melanogaster species group. A mode of evolution for this gene family in Drosophila is proposed.

Repetitive arrays containing a housekeeping gene have altered polytene chromosome morphology in Drosophila.

Much of our understanding of gene and chromatin organization has been developed from observation of polytene chromosomes. We describe an experimental approach using transgenes that has allowed us to observe local changes in polytene morphology. A composite P transposon that contains a fusion between the regulatory region of Prat, a purine synthesis gene, and brown (bw), an eye pigment reporter, was transformed into the 65A10 polytene band and subjected to P-transposase mutagenesis. Arrays of up to 320 kb at 65A10 were recovered by selection for increased pigment, and pigment levels were found to be proportional to numbers of copies. In polytene chromosomes, the original transformant was found to split 65A10 into two thin bands separated by an interband. With increases in copy number, the interband became progressively denser, eventually forming a dark, amorphous, deformable structure unlike any previously reported. The persistence of Prat expression in development, together with the cytological appearance of these large arrays, suggest that the state of the Prat promoter is affecting polytene structure. Because this unique structure is distinct from bands, interbands, puffs, and the chromocenter, which comprise polytene chromosomes, we suggest that it is composed of an altered form of chromatin.

Alpha-adaptin, a marker for endocytosis, is expressed in complex patterns during Drosophila development.

A Drosophila cDNA encoding a structural homologue of the mammalian coated vesicle component alpha-adaptin (AP2 adaptor complex) has been cloned and sequenced. The mammalian and invertebrate sequences are highly conserved, especially within the amino terminal region, a domain that mediates interactions with other components within the AP2 complex and with specific receptors tails. Mammalian alpha-adaptins are encoded by two genes; however, Drosophila alpha-adaptin has a single gene locus, within polytene bands 21C2-C3 on the left arm of the chromosome 2, closely adjacent to the paired homeobox gene aristaless. There seem to be at least two Drosophila alpha-adaptin transcripts expressed, plausibly by alternative splicing. One of the transcripts is more abundant during early embryogenesis and may be of maternal origin. We have studied the distribution of the alpha-adaptin protein throughout embryogenesis and at the neuromuscular junction of the third instar larva. During cellularization of the blastoderm embryo, the protein is seen between and ahead of the elongating nuclei, and then redistributes to the cell surface during gastrulation. These observations suggest a role for endocytosis in cellularization and are consistent with the finding that dynamin (the shibire gene product), another component of the endocytic mechanism, is required for cellularization. At later stages of embryogenesis, alpha-adaptin is expressed in complex and dynamic patterns. It is strongly induced in elements of the central and peripheral nervous system (e.g., in neuroblasts, the presumptive stomatogastric nervous system, and the lateral chordotonal sense organs), in the Garland cells, the adult midgut precursors, the antenno-maxillary complex, the endoderm, the fat bodies, and the visceral mesoderm. In the larva, alpha-adaptin is localized at the plasma membrane in the synaptic boutons of the neuromuscular junctions. The cells expressing high levels of alpha-adaptin are known or expected to support high levels of endocytosis; thus, this coated vesicle protein seems to be an excellent marker for endocytic activity. The expression patterns of dynamin, detected in the embryo by in situ hybridization methods, are very similar to those reported here for alpha-adaptin reflecting the likely coordinated expression of endocytic components. Taken together with previous evidence, our results suggest that endosomal vesicle trafficking, membrane recycling, and the regulation of endocytosis play critical roles in the wide range of developmental processes.

Drosophila melanogaster contains both X-linked and autosomal homologues of the gene encoding calcineurin B

A transcription unit was identified in the 43E polytene band region of the second chromosome of Drosophila melanogaster (Dm) whose putative translation product has 85% amino acid (aa) identity with the B subunit of the calcineurin protein (CnB) from humans. Unlike the previously described intronless Dm CnB gene homologue, which is located within the 4F band region of the X chromosome, the coding region of this second CnB is found to be interrupted by three introns. Conceptual translation of both Dm CnB genes predict proteins of identical size that are 98% identical in aa sequence. Northern blot analyses indicate that Dm pupae and adults express two different CnB-encoding transcripts that are differentially regulated.

Molecular cytogenetic organization of polytene chromosomes

The results of the works carried out in the Laboratory of Molecular Cytogenetics (Institute of Cytology and Genetics of Siberian Branch of the RAS, Novosibirsk) devoted to the molecular genetic analysis of main units of polytene chromosomes, bands, interbands, and puffs, as well as intercalary and pericentric heterochromatin, are summarized. The results are discussed in terms of the dynamic model of organization of polytene chromosomes.

Structure and regulation of a gene cluster for male accessory gland transcripts in Drosophila melanogaster

We characterize a cluster of three genes, named Mst57Da, Mst57Db and Mst57Dc, according to their localization at polytene band 57D and their selective expression in Drosophila melanogaster males. Within a 4 kb segment of genomic DNA these genes code for poly(A) + RNAs of about 750 nt, 650 nt and 850 nt length that are restricted to the male accessory glands. They all contain relatively long 5′ and 3′ untranslated regions compared to the size of their open reading frames. The 5′ untranslated regions are characterized by several short open reading frames. In addition, the genes Mst57Da and Mst57Db share a 14 nucleotides long conserved sequence block in front of the putative translated regions. We propose that these common features are structural elements important for translational control of these genes. The predicted sequences of the small proteins of 55, 40 and 42 amino acids suggest that two of them ( MST57Da) and ( MST57Db) are secretory proteins and therefore components of the paragonial secretions. In P-element mediated germ line transformations of Mst57Db/lacZ fusion genes, a short gene fragment of −187/+ 395 is still specifically expressed in the paragonia. In addition, regions involved in negative and positive control of transcription have been identified. Development of a supernumerary paragonial gland is frequently observed in transgenic, intersexual genotypes indicating that in intersexes cells derived from the female genital disc can develop male genital structures.

Recovery of dominant, autosomal flightless mutants of Drosophila melanogaster and identification of a new gene required for normal muscle structure and function.

To identify further mutations affecting muscle function and development in Drosophila melanogaster we recovered 22 autosomal dominant flightless mutations. From these we have isolated eight viable and lethal alleles of the muscle myosin heavy chain gene, and seven viable alleles of the indirect flight muscle (IFM)-specific Act88F actin gene. The Mhc mutations display a variety of phenotypic effects, ranging from reductions in myosin heavy chain content in the indirect flight muscles only, to reductions in the levels of this protein in other muscles. The Act88F mutations range from those which produce no stable actin and have severely abnormal myofibrillar structure, to those which accumulate apparently normal levels of actin in the flight muscles but which still have abnormal myofibrils and fly very poorly. We also recovered two recessive flightless mutants on the third chromosome. The remaining five dominant flightless mutations are all lethal alleles of a gene named lethal(3)Laker. The Laker alleles have been characterized and the gene located in polytene bands 62A10,B1-62B2,4. Laker is a previously unidentified locus which is haplo-insufficient for flight. In addition, adult wild-type heterozygotes and the lethal larval trans-heterozygotes show abnormalities of muscle structure indicating that the Laker gene product is an important component of muscle.

A yeast artificial chromosome clone map of the Drosophila genome.

We describe the mapping of 979 randomly selected large yeast artificial chromosome (YAC) clones of Drosophila DNA by in situ hybridization to polytene chromosomes. Eight hundred and fifty-five of the clones are euchromatic and have primary hybridization sites in the banded portions of the polytene chromosomes, whereas 124 are heterochromatic and label the chromocenter. The average euchromatic clone contains about 211 kb and, at its primary site, labels eight or nine contiguous polytene bands. Thus, the extent as well as chromosomal position of each clone has been determined. By direct band counts, we estimate our clones provide about 76% coverage of the euchromatin of the major autosomes, and 63% coverage of the X. When previously reported YAC mapping data are combined with ours, euchromatic coverage is extended to about 90% for the autosomes and 82% for the X. The distribution of gap sizes in our map and the coverage achieved are in good agreement with expectations based on the assumption of random coverage, indicating that euchromatic clones are essentially randomly distributed. However, certain gaps in coverage, including the entire fourth chromosome euchromatin, may be significant. Heterochromatic sequences are underrepresented among the YAC clones by two to three fold. This may result, at least in part, from underrepresentation of heterochromatic sequences in adult DNA (the source of most of the clones analyzed), or from clone instability.

Polytene chromosomes of monogenic and amphogenic Chrysomya species (Calliphoridae, Diptera): analysis of banding patterns and in situ hybridization with Drosophila sex determining gene sequences.

Standard maps for the five banded polytene chromosomes found in trichogen cell nuclei of the monogenic blowfly Chrysomya rufifacies and the amphogenic Chrysomya pinguis are presented. The chromosomes are highly homologous in the two species; differences in banding patterns are predominantly caused by one pericentric and ten paracentric inversions. In chromosome 5 of the amphogenic Chrysomya phaonis, also analysed in this paper, an additional paracentric inversion was observed. The distribution of species specific inversions indicates that the monogenic C. rufifacies is phylogenetically older than the amphogenic species. The maternal sex realizer locus F'/f on polytene chromosome 5 of C. rufifacies is not associated with a structural heterozygosity. Chromosome pair 6 of C. rufifacies and the sex chromosome pair of C. pinguis are under-replicated in polytene nuclei; they consist of irregular chromatin granules, frequently associated with nucleolus material. Evolution of heteromorphic sex chromosomes in Chrysomya is probably correlated with heterochromatin accumulation. A search for sex determining genes in Chrysomya was initiated using sex determining sequences from Drosophila melanogaster for in situ hybridization. The polytene band 41A1 on chromosome 5 of monogenic and amphogenic Chrysomya species contains sequences homologous to the maternal sex determining gene daughterless (da). Homology to the zygotic gene Sex-lethal (Sxl) of Drosophila is detected in band 39A1 on chromosome 5 of C. rufifacies. The findings reported here are the first evidence for a possible homology between the da gene of Drosophila and the maternal sex realizer F' of C. rufifacies. An hypothesis for the evolution of the maternal effect sex determination of C. rufifacies is proposed.

Molecular and genetic analyses of Drosophila Prat, which encodes the first enzyme of de novo purine biosynthesis.

The Drosophila Prat gene encodes phosphoribosylamidotransferase (PRAT), the enzyme that performs the first committed step of the de novo purine nucleotide biosynthesis pathway. Using information from amino acid sequence alignments of PRAT from other organisms, a polymerase chain reaction-based approach was employed to clone Prat. Amino acid sequence alignment of Drosophila PRAT with PRAT from bacteria, yeast, and vertebrates indicates that it is most identical (at least 60%) to the vertebrate PRATs. It shares putative amino-terminal propeptide and iron-binding domains seen only in Bacillus subtilis and vertebrate PRATs. Prat was localized to the right arm of chromosome 3 at polytene band 84E1-2. Owing to the fact that this region had been well characterized previously, Prat was localized to a 30-kilobase region between two deficiency breakpoints. By making the prediction that Prat would have a similar "purine syndrome" phenotype as mutations in the genes ade2 and ade3, which encode enzymes downstream in the pathway, five alleles of Prat were isolated. Three of the alleles were identified as missense mutations. A comparison of PRAT enzyme activity with phenotype in three of the mutants indicates that a reduction to 40% of the wild-type allele's activity is sufficient to cause the purine syndrome, suggesting that PRAT activity is limiting in Drosophila.

Comparative analysis of the sequence and structure of two Drosophila melanogaster genes encoding vitelline membrane proteins

Two Drosophila melanogaster vitelline membrane protein-encoding genes ( VM), located at polytene band positions 26A and 34C, have been cloned and comparatively characterized at the nucleotide level. Sequence analysis of genomic and cDNA clones for the two genes, VM26A.1 and VM34C.1, indicates that both are similarly organized with a central highly conserved domain [Scherer et al., Dev. Biol. 130 (1988) 786–788] which is flanked by unrelated regions, and that both genes lack introns. Comparison of the upstream regions reveals that both VM genes contain a heptameric element identical to one associated with the D. melanogaster yolk protein-encoding genes ( YP). This heptamer occurs in the specific 5' flanking region responsible for ovarian temporal- and tissue-specific control in both VM and YP genes. A putative chorion transcription factor 2 site is also associated with an upstream control element of VM26A.1, but not with any sequenced portion of VM34C.1.