Polytene Chromosome Puffs Research Articles

Evidence against a Role for the JIL-1 Kinase in H3S28 Phosphorylation and 14-3-3 Recruitment to Active Genes in Drosophila

JIL-1 is the major kinase controlling phosphorylation of histone H3S10 and has been demonstrated to function to counteract heterochromatization and gene silencing. However, an alternative model has been proposed in which JIL-1 is required for transcription to occur, additionally phosphorylates H3S28, and recruits 14-3-3 to active genes. Since these findings are incompatible with our previous demonstration that there are robust levels of transcription in the complete absence of JIL-1 and that JIL-1 is not present at developmental or heat shock-induced polytene chromosome puffs, we have reexamined JIL-1’s possible role in H3S28 phosphorylation and 14-3-3 recruitment. Using two different H3S28ph antibodies we show by immunocytochemistry and immunoblotting that in Drosophila the H3S28ph mark is not present at detectable levels above background on polytene chromosomes at interphase but only on chromosomes at pro-, meta-, and anaphase during cell division in S2 cells and third instar larval neuroblasts. Moreover, this mitotic H3S28ph signal is also present in a JIL-1 null mutant background at undiminished levels suggesting that JIL-1 is not the mitotic H3S28ph kinase. We also demonstrate that H3S28ph is not enriched at heat shock puffs. Using two different pan-specific 14-3-3 antibodies as well as an enhancer trap 14-3-3ε-GFP line we show that 14-3-3, while present in salivary gland nuclei, does not localize to chromosomes but only to the nuclear matrix surrounding the chromosomes. In our hands 14-3-3 is not recruited to developmental or heat shock puffs. Furthermore, using a lacO repeat tethering system to target LacI-JIL-1 to ectopic sites on polytene chromosomes we show that only H3S10ph is present and upregulated at such sites, not H3S28ph or 14-3-3. Thus, our results argue strongly against a model where JIL-1 is required for H3S28 phosphorylation and 14-3-3 recruitment at active genes.

Heat Shock Proteins: An Alternative to Control Disease in Aquatic Organism

The heat shock response was discovered by Ritossa and co-workers in 1962 when the exposure of Drosophila larvae to elevate temperature was observed to generate salivary gland polytene chromosome puffs, subsequently proven to reflect transcriptional induction of heat shock protein (Hsp) genes. Hsps, also referred to as stress proteins and molecular chaperones, normally account for 5-10% of total cellular protein, and they increasetwo or three times when cells are exposed to biotic and abiotic stressors [1]. Hsps occur in all living organism and they are categorized into several families based on function, sequence and molecular mass. Major Hsp families’ include Hsp110, Hsp100, Hsp 90, Hsp70, Hsp 60, and the small Hsps (sHsps), with the latter having a molecular mass less than 40 kDa. Constitutive and inducible Hsps, some of which are organelle specific, perform vital functions generally by interacting with exposed hydrophobic surfaces of nascent and stress-induced non-native proteins [2]. Hsps help other proteins find their correct three-dimensional configurations by keeping them in folding-competent, folded, or unfolded states assist protein localization to specific organelles, and target non-native or aggregated proteins for degradation and removal from the cell. In addition to stress resistance, Hsps are involved in animal and plant development, aging, environmental adaptation, and the immune response, demonstrating the fundamental importance of these proteins to cell survival [3].

Comparing active and repressed expression states of genes controlled by the Polycomb/Trithorax group proteins

Drosophila Polycomb group (PcG) and Trithorax group (TrxG) proteins are responsible for the maintenance of stable transcription patterns of many developmental regulators, such as the homeotic genes. We have used ChIP-on-chip to compare the distribution of several PcG/TrxG proteins, as well as histone modifications in active and repressed genes across the two homeotic complexes ANT-C and BX-C. Our data indicate the colocalization of the Polycomb repressive complex 1 (PRC1) with Trx and the DNA binding protein Pleiohomeotic (Pho) at discrete sequence elements as well as significant chromatin assembly differences in active and inactive regions. Trx binds to the promoters of active genes and noncoding transcripts. Most strikingly, in the active state, Pho covers extended chromatin domains over many kilobases. This feature of Pho, observed on many polytene chromosome puffs, reflects a previously undescribed function. At the hsp70 gene, we demonstrate in mutants that Pho is required for transcriptional recovery after heat shock. Besides its presumptive function in recruiting PcG complexes to their site of action, our results now uncover that Pho plays an additional role in the repression of already induced genes.

Temperature-induced alteration of the polytene X chromosome structure in male larvae of the strain In(1)BM2 (reinverted) of Drosophila melanogaster.

In Drosophila melanogaster, the polytene X chromosome of male third instar larva appears twice as wide as an unpaired female X chromosome or an autosome. This characteristic morphology of the male X chromosome is correlated with the increased rate of transcription of the sex-linked genes, which ensures gene dosage compensation. In male third instar larvae of the strain In(1)BM2 (reinverted), polytene nuclei manifest unusually puffy X chromosomes at 18 +/- 1 degrees C. Such 'puffy X' chromosomes are pompons, that is, despite the increased width of the chromosome, transcription remains at the wild-type level. This characteristic is a caveat to the invariable correlation between polytene chromosome puffs and transcription, and suggests that the mutant X chromosomes arise due to perturbation of a pathway that controls the structure but not the transcription of the polytene X chromosome. In this report we present evidence that the pompons of In(1)BM2 (reinverted) arise due to spiralization of the male X chromosome, which results in condensing of the chromosome. This unusual structural alteration can be induced only in male larvae of this strain, at the third instar larval stage, through temperature shifts from 24 +/- 1 degrees C to 18 +/- 1 degrees C and during recovery from cold shock. Furthermore, extract from male adult, pupae and third instar larvae can induce chromosome condensation in wild-type larvae in vitro. This new evidence not only explains the absence of correlation between chromosome width and transcription of the pompons of In(1)BM2 (reinverted), but also suggests that the chromosomal rearrangement perturbs a pathway that regulates the condensation of chromosomes.

The L63 Gene Is Necessary for the Ecdysone-Induced 63E Late Puff and Encodes CDK Proteins Required for Drosophila Development

The pulse of ecdysone that triggers Drosophila metamorphosis activates six early genes in a primary response made visible by polytene chromosome puffs. The secondary response is detected by the induction of over 100 late puffs, only a few of which have been subject to molecular genetic analysis. We present a molecular and mutational analysis of the L63 gene responsible for the late puff at 63E. This gene contains overlapping L63A, B, and C transcription units of which the A unit encodes two isoforms and the B unit three. The C unit, which exhibits little activity, encodes one of the B isoforms. Evidence that L63B, but not L63A, transcription is ecdysone responsive derives from their developmental transcription profiles and from P-element mutagenesis showing that ecdysone induction of the 63E puff requires sequences adjacent to the 5′ end of L63B but not those adjacent to the 5′ end of L63A. L63-specific lethal mutations showed that L63 is required not only for metamorphosis, but also maternally and for embryonic and larval development. The L63 proteins contain a common C-terminal 294-aa sequence that is 71% identical to the CDK sequence of the murine PFTAIRE protein. In vivo tests of L63 proteins altered by site-directed mutagenesis showed that they exhibit CDK functions. L63 proteins are widely distributed among late larval and prepupal tissues and are unlikely to be involved in cell cycle functions.

DEVELOPMENTAL REGULATION OF GRANULE SIZE AND NUMBERS IN LARVAL SALIVARY GLANDS OF DROSOPHILA BY STEROID HORMONE ECDYSONE

The size and number of secretory granules in late larval salivary glands of Drosophila melanogaster have been related to interecdysial and early metamorphic development represented by well-known puffs in polytene chromosomes. Interecdysial period (puff stage 1 (PS1)) is characterized by presence of numerous small granules (11,000 per cell). The transition from PSI to early metamorphic phase (PS2 and upwards), induced by rapid elevation in endogenous steroid hormone ecdysone, is accompanied by continuous growth of granule diameter with concomitant reduction in their number per cell. In the PS4, just prior to secretion, ∼3000 mature granules occur per cell. The mature state is associated with the change from hyperbolic to Gaussian distribution of granule number over their size range. Similar changes in secretory granule parameters were observed in interecdysial salivary glands explanted from 3rd instar larvae and cultured in vitro in medium containing 5×10−6m ecdysone.

Sodium Salicylate Decreases Intracellular ATP, Induces Both Heat Shock Factor Binding and Chromosomal Puffing, but Does Not Induce hsp 70 Gene Transcription in Drosophila

Sodium salicylate has long been known to be an inducer of the heat shock puffs and presumably heat shock gene transcription in the polytene chromosomes of Drosophila salivary gland cells. Stress-induced transcription of the heat shock genes is mediated by the transcription factor known as Heat Shock Factor (HSF). In yeast, sodium salicylate has been reported to induce the DNA binding of HSF but not heat shock gene transcription itself, and similar findings have been reported in human cells. This apparent discrepancy in the induction of certain aspects of the heat shock response between these organisms prompted us to carefully reexamine the induction of the heat shock response in Drosophila salivary gland cells of third instar larvae and Drosophila tissue culture (SL2) cells. Sodium salicylate (3-30 mM) decreases intracellular ATP levels in SL2 cells and induces HSF binding activity in SL2 and salivary gland cells in a dose-dependent manner. Despite the induction of HSF binding and heat shock puffs in polytene chromosomes, we found no evidence for increased hsp 70 gene transcription suggesting that chromosomal puffing and gene transcription may be separable events. Salicylate did not induce the HSF hyperphosphorylation that is normally associated with HSF activation. Furthermore, salicylate (30 mM) prevented heat-induced hyperphosphorylation of HSF and hsp 70 gene transcription indicating that salicylate's inhibitory effect on hsp 70 transcription may be independent of its effect on HSF binding activity. We propose that the reduction in intracellular ATP caused by the addition of salicylate likely plays a role in the activation of HSF binding and the inhibition of both HSF hyperphosphorylation and hsp 70 gene transcription.

Telomeric puffing induced by heat shock inChironomus thummi

We summarize the most remarkable features of the heat shock inducible large telomeric puffs (T-BRs) in polytene chromosomes of Chironomus thummi. Kinetic aspects of formation of T-BRs as well as their transcriptional behaviour clearly support the view that T-BRs are components of the heat shock response in Chironomus. Available molecular data indicate T-BRs to include long arrays of 176 bp tandem repeats. A large transcript (> 10 kb) encompas- sing the telomere associated repeat has been detected. Several other similarities between T-BRs of Chironomus and the hsrω genes of Drosophila suggest the T-BRs to be hsrω counterpart in Chironomus.

The Drosophila E93 Gene from the 93F Early Puff Displays Stage- and Tissue-Specific Regulation by 20-Hydroxyecdysone

Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, we have isolated and characterized the E93 gene responsible for the stage-specific 93F early puff. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. E93 spans at least 55 kb of genomic DNA and encodes a 146-kDa protein that has no matches in the sequence databases, but displays several characteristics of known Drosophila transcription factors. We propose that E93 acts in a stage-specific regulatory hierarchy in the salivary gland to direct its histolysis in response to the prepupal ecdysteroid pulse.

The Drosophila 63F early puff contains E63-1, an ecdysone-inducible gene that encodes a novel Ca2+-binding protein

Pulses of ecdysone at the end of Drosophila larval development dramatically reprogram gene expression as they signal the onset of metamorphosis. Ecdysone directly induces several early puffs in the salivary gland polytene chromosomes that, in turn, activate many late puffs. Three early puffs, at 2B5, 74EF, and 75B, have been studied at the molecular level. Each contains a single ecdysone primary-response gene that encodes a family of widely expressed transcription factors. We report here a molecular characterization of the 63F early puff. Unexpectedly, we have found this locus to be significantly different from the previously characterized early puff loci. First, the 63F puff contains a pair of ecdysone-inducible genes that are transcribed in the larval salivary glands: E63-1 and E63-2. Second, E63-1 induction in late third instar larvae appears to be highly tissue-specific, restricted to the salivary gland. Third, E63-1 encodes a novel Ca(2+)-binding protein related to calmodulin. The discovery of an ecdysone-inducible Ca(2+)-binding protein provides a foundation for integrating steroid hormone and calcium second messenger signaling pathways and generates an additional level for potential regulation of the ecdysone response.

The Drosophila E74 gene is required for the proper stage- and tissue-specific transcription of ecdysone-regulated genes at the onset of metamorphosis.

The steroid hormone ecdysone directly induces a small set of early genes, visible as puffs in the larval salivary gland polytene chromosomes, as it signals the onset of Drosophila metamorphorsis. The products of these genes appear to function as regulators that both repress their own expression and induce a large set of secondary-response late genes. We have identified recessive loss-of-function mutations in the early gene E74, a member of the ets protooncogene family that encodes two related DNA-binding proteins, E74A and E74B. These mutations cause defects in pupariation and pupation, and result in lethality during metamorphosis. Here we extend our phenotypic characterization of the E74A and E74B mutant alleles to the molecular level by examining their effects on the transcription of over 30 ecdysone-regulated genes. We show that the transcription of most ecdysone primary-response genes during late larval and prepupal development is unaffected by the E74 mutations. Rather, we find that E74 is necessary for the appropriate regulation of many ecdysone secondary-response genes. E74B is required for the maximal induction of glue genes in mid third instar larval salivary glands, while E74A is required in early prepupae for the proper timing and maximal induction of a subset of late genes. E74 activity is also necessary for the correct regulation of genes expressed predominantly in the fat body, epidermis or imaginal discs. These observations confirm that E74 plays a critical role in regulating transcription during the early stages of Drosophila metamorphosis. In addition, the widespread effects of the E74 mutations on transcription indicate that E74 functions in regulatory hierarchies not only in the larval salivary gland, but throughout the entire organism.

Sequential gene activation by ecdysone in Drosophila melanogaster: the hierarchical equivalence of early and early late genes

Ecdysteroids are key regulators of insect development. In Drosophila melanogaster the late larval response to ecdysone is characterised by a precise sequential activation of members of the superfamily of nuclear receptors (DHR3, DHR39, EcR, E75, E78, FTZ-F1, usp). Many of these genes are localised in the polytene chromosome puffs of the salivary gland previously classified as intermoult, early or early-late puff loci. Ashburner et al. (Ashburner, M., Chihara, C., Meltzer, P. and Richards, G. (1974) Cold Spring Harbour Symp. Quant. Biol. 38, 655-662) proposed a formal model describing interactions between ecdysone, its receptor and the early and late puffs during this ecdysone response. To integrate transcripts from the intermoult and early-late puffs into this model, we have used a micro RT-PCR assay to study their hormonal regulation using salivary gland culture protocols first used in the puffing analyses. We show that transcripts from certain early-late puffs are induced in parallel with the early transcripts and are thus hierarchically equivalent. In vivo the profile of the increase in hormone titre, the sensitivity of different promoters to hormone and the rate of transcript accumulation must contribute to the temporal differences in expression observed between these two classes.

A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone

Two successive pulses of ecdysone signal the ends of larval and prepupal development in Drosophila, inducing early and late puffs in the salivary gland polytene chromosomes. Early puff induction in prepupae is dependent on a preceding period of protein synthesis and low ecdysone concentration. We demonstrate here that the competence acquired during this interval can be provided by βFTZ-F1, a nuclear hormone receptor superfamily member derived from the 75CD midprepupal puff. We show that βFTZ-F1 represses its own transcription and is repressed by ecdysone, explaining its bried expression in mid-prepupae. We further show that ectopic βFTZ-F1 expression leads to enhanced levels of ecdysone-induced BR-C, E74, and E75 early gene transcription and premature induction of the stage-specific 93F early puff and E93 transcription. These findings indicate that βFTZ-F1 plays a central role in the prepupal genetic response to ecdysone and provide a molecular mechanism for stage-specific responses to steroid hormones.

Isolation and characterization of fifteen ecdysone-inducible Drosophila genes reveal unexpected complexities in ecdysone regulation.

Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.

Isolation and Characterization of Fifteen Ecdysone-Inducible Drosophila Genes Reveal Unexpected Complexities in Ecdysone Regulation

Our insights into the regulatory mechanisms by which the steroid hormone ecdysone triggers Drosophila melanogaster metamorphosis have largely depended on puffs in the larval salivary gland polytene chromosomes as a means of identifying genes of interest. Here, we describe an approach that provides access to ecdysone-inducible genes that are expressed in most larval and imaginal tissues, regardless of their ability to form puffs in the polytene chromosomes. Several hundred cDNAs were picked at random from subtracted cDNA libraries and subjected to a rapid and sensitive screen for their ability to detect mRNAs induced by ecdysone in the presence of cycloheximide. Of the 15 genes identified in this manner, 2 correspond to early puffs in the salivary gland polytene chromosomes, at 63F and 75B, confirming that this screen functions at the desired level of sensitivity and is capable of identifying novel primary-response genes. Three of the genes, Eig45-1, Eig58, and Eig87, are expressed coordinately with the salivary gland early genes; one of them, Eig58, maps to the 58BC puff that is active when the 74EF and 75B early puffs are at their maximal size. Another gene identified in this screen, Eig17-1, encodes a novel cytochrome P-450. On the basis of its sequence identity and temporal profile of expression, this gene may play a role in steroid hormone metabolism and thus could provide a mechanism for feedback regulation of ecdysone production. Although all 15 genes have patterns of transcription that are consistent with ecdysone regulation in vivo, 5 genes do not appear to be induced by the late larval ecdysone pulse. This indicates that ecdysone induction in larval organs cultured with cycloheximide is not always indicative of a primary response to the hormone.

The reaction with polytene chromosomes of antibodies raised against Drosophila E75A protein

The steroid insect molting hormone ecdysone rapidly induces a small number of polytene chromosome puffs in Drosophila. The Ashburner model proposes that the corresponding early genes encode proteins involved in both the induction of the late genes and the repression of the early genes. The Drosophila E75 early gene has been isolated and two of its products, E75A and E75B, have been shown to be members of the steroid receptor superfamily. We have now prepared antisera directed against A- and B-specific regions of the E75 proteins. Antisera and a monoclonal antibody raised against E75A, the major larval protein product of the E75 gene, bind to discrete sites in native salivary gland chromosomes. These sites are closely correlated with early and late ecdysone responsive loci.

Isolation and characterization of five Drosophila genes that encode an ets-related DNA binding domain

The recent determination of the site-specific DNA binding properties of several proteins related to the ets oncoprotein has allowed the definition of a novel DNA binding domain, designated the ETS domain. In Drosophila, an ETS domain is present in the early ecdysone-induced E74A protein, which binds DNA in a site-specific manner and interacts with many ecdysone-induced polytene chromosome puffs at the onset of metamorphosis. As a first step toward determining the function of ETS-domain proteins during Drosophila development, we have used PCR amplification with degenerate oligonucleotides to isolate five other ets-related genes. Two of these genes, D- ets-2 and D- elg, have been previously identified. The proteins encoded by these genes are highly related to one another and to the seven identified vertebrate ETS-domain proteins, within the ∼85-amino-acid DNA binding domain. In situ hybridization to polytene chromosomes revealed that these ets-related genes are not clustered in the genome and that only E74 corresponds to an ecdysone-inducible puff locus. These five ets-related genes are distinguished further from E74 in that they are transcribed through most of development, suggesting that they do not perform a stage-specific function. They are, however, expressed in a variety of patterns in early embryos, suggesting roles in the development of specific cell types. D- ets-2 is expressed in a complex pattern that changes dynamically during early embryogenesis. D- ets-3 and D- ets-6 are expressed in the ventral nervous system. The expression of D- ets-3 is higher in the three thoracic segments and lower in the abdominal segments. The high levels of expression in the thoracic segments are dependent on the presence of the bithorax complex. D- ets-4 and D- elg are expressed at their highest levels in the pole cells, suggesting a role in the development of the germline. This study represents the first effort in any organism to systematically isolate members of the ets gene family. The identification of six independent ets-related genes demonstrates that the ETS-domain proteins constitute a new family of potential transcriptional regulators encoded by the Drosophila genome.

Patterns of E74A RNA and protein expression at the onset of metamorphosis in Drosophila

Metamorphosis in Drosophila is triggered by a pulse of the steroid hormone ecdysone at the end of larval development. Ecdysone initiates a genetic hierarchy that can be visualized as a series of puffs in the larval salivary gland polytene chromosomes. The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins which appear to play a regulatory role in the hierarchy. Here we describe the spatial and temporal patterns of E74A RNA and protein expression at the onset of metamorphosis. We use in situ hybridization, antibody stains, and western and northern blot analyses to follow E74A expression from its initial appearance as nascent transcripts on the polytene chromosomes, to spliced mRNA, to post-translationally modified nuclear E74A protein. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74A RNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. The spatial distribution of nuclear E74A protein correlates with the RNA distribution with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen in 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. We discuss possible mechanisms for post-transcriptional regulation of E74A expression and suggest that the unusually long and complex 5' leader in the E74A mRNA may regulate its translation.

Interspecific comparisons of the structure and regulation of the Drosophila ecdysone-inducible gene E74.

The Drosophila melanogaster E74 gene is induced directly by the steroid hormone ecdysone and is a member of a small set of "early" genes that appear to trigger the onset of metamorphosis. The gene consists of three overlapping transcription units encoding two proteins, E74A and E74B, which possess a common C terminus. According to the Ashburner model for ecdysone's action, an E74 protein product potentially functions as a transcriptional activator of "late" genes as well as a repressor of early genes. We have taken an evolutionary approach to understand the function and regulation of E74 by isolating the homologous genes from Drosophila pseudoobscura and Drosophila virilis and comparing them to D. melanogaster E74 sequences. Conserved characteristics of the E74 genes include ecdysone inducibility, localization to ecdysone-induced polytene chromosome puffs, and gene size. Amino acid sequence comparisons of the E74A protein reveal a highly conserved C-terminal region that is rich in basic amino acid residues and which has been proposed to possess sequence-specific DNA binding activity. The moderately conserved N-terminal region has maintained its overall acidic character and is a potential transcriptional activator domain. The central region contains conserved glutamine and alanine homopolymeric repeats of variable lengths. Nucleotide sequence comparisons of the E74A promoter region fail to reveal ecdysone-response elements but do identify conserved sequences that may function in E74A regulation.

DrosophilasnRNP associated protein P11 which specifically binds to heat shock puff 93D reveals strong homology with hnRNP core protein A1

We have isolated cDNAs coding for a ribonucleoprotein of Drosophila melanogaster that is distinguished by its nearly exclusive presence at only one of the several heat shock puffs in polytene chromosomes of third instar larvae. We determined the nucleotide sequence and deduced the corresponding amino acid sequence. Its coding capacity for a 39 kDa protein is consistent with the size of the protein detected by the monoclonal antibody P11 used for expression cloning. Our results show that the P11 protein belongs to the category of hnRNP proteins of bipartite structure: the amino-terminal half contains two RNA binding domains and the carboxyterminal half is rich in glycine residues. Analysis of the genomic structure revealed two introns located within the coding portion of the gene and a third one in the 3'untranslated region. We detect two different polyadenylation sites as a result of alternative termination-polyadenylation. Its strong sequence homology with hnRNP A1 protein and its previously shown association with snRNP particles indicates that a typical hnRNP protein may also exist in a complex with snRNP particles. The P11 sequence corresponds to the Hrb87F sequence that was recently described by Haynes et al. (1) as hnRNP A related gene.