Isolation of mRNA from specific tissues of Drosophila by mRNA tagging

To study the function of specific cells or tissues using genomic tools like microarray analyses, it is highly desirable to obtain mRNA from a homogeneous source. However, this is particularly challenging for small organisms, like Caenorhabditis elegans and Drosophila melanogaster. We have optimized and applied a new technique, mRNA tagging, to isolate mRNA from specific tissues of D.melanogaster. A FLAG-tagged poly(A)-binding protein (PABP) is expressed in a specific tissue and mRNA from that tissue is thus tagged by the recombinant PABP and separated from mRNA in other tissues by co-immunoprecipitation with a FLAG-tag specific antibody. The fractionated mRNA is then amplified and used as probe in microarray experiments. As a test system, we employed the procedures to identify genes expressed in Drosophila photoreceptor cells. We found that most known photoreceptor cell-specific mRNAs were identified by mRNA tagging. Furthermore, at least 11 novel genes have been identified as enriched in photoreceptor cells. mRNA tagging is a powerful general method for profiling gene expression in specific tissues and for identifying tissue-specific genes.     

The ribosomal DNA of Drosophila melanogaster is organized differently from that of Drosophila hydei

On the X chromosome of Drosophila melanogaster there is a single tandem array of 240 ribosomal RNA genes. The majority of these contain an insertion, known as type I, in the 28 S coding region. Previous genetic and electron microscopic studies indicated that genes bearing the type I insertion (ins⁺) are interspersed at random with those lacking it (ins^?). In contrast, Renkawitz-Pohl et al. (1981) have analyzed the restriction pattern of X chromosomal ribosomal DNA in Drosophila hydei and demonstrated that in this case ins⁺ genes are segregated from ins^?. This suggests either that the rDNA is organized differently in these two species or that the restriction enzyme technique reveals significant clustering not detected by previous methods. By using an appropriate restriction enzyme, we demonstrate that ins⁺ and ins^? genes are intermingled at random in D. melanogaster. These experiments also indicate that genes containing the short form of the insertion are flanked by a larger spacer upstream than downstream.     

PTC and PROP behave differently in tests of discrimination from their solvents

Better discrimination was possible between phenylthiocarbamide (PTC) solutions and the pure solvent when the solvent was a tasteless low- concentration NaCl solution to which the subject had adapted than when the solvent was purified water. The reverse was true for 6-n- propylthiouracil (PROP). The differences in discrimination for PROP and PTC in the different solvents were caused by differences in the intensity and persistence of aftertastes, rather than a more intense perception of the PTC and PROP tastes per se. This has consequences for traditional approaches to measuring taste sensitivity, as well as indicating that PTC and PROP are not necessarily equivalent indicators of 'taster' versus 'non-taster' status.      

Multiple circadian-regulated elements contribute to cycling period gene expression in Drosophila.

A new regulatory element necessary for the correct temporal expression of the period (per) gene was identified by monitoring real-time per expression in living individual flies carrying two different period-luciferase transgenes. luciferase RNA driven from only the per promoter was not sufficient to replicate the normal pattern of per RNA cycling; however, a per-luc fusion RNA driven from a transgene containing additional per sequences cycled identically to endogenous per. The results indicate the existence of at least two circadian-regulated elements--one within the promoter and one within the transcribed portion of the per gene. Phase and amplitude analysis of both per-luc transgenes revealed that normal per expression requires the regulation of these elements at distinct phases and suggests a mechanism by which biological clocks sustain high-amplitude feedback oscillations.     

ATPase-defective derivatives of Escherichia coli DnaK that behave differently with respect to ATP-induced conformational change and peptide release.

We have characterized the effects of the T199S, T199A, and K70A mutations on the biochemical activity and in vivo functioning of Escherichia coli DnaK. Threonine-199 is the site of autophosphorylation of DnaK, and the lysine residue of bovine Hsc70 corresponding to K70 of DnaK has been shown to be essential for the hydrolysis of ATP. The dnaK alleles T199A and K70A are completely unable, and the T199S allele is only partially able, to complement the defects of a DeltadnaK mutant. The ATPase activities of the DnaK T199A and DnaK K70A proteins are nearly abolished, while the ATPase activity of the DnaK T199S protein has a steady-state rate similar to that of wild-type DnaK. The DnaK T199S protein also retains approximately 13% of the autophosphorylation activity of wild-type DnaK, while the autophosphorylation activities of the T199A and K70A derivatives are completely abolished. All four DnaK proteins bind a model peptide substrate, and the wild-type, T199A, and T199S DnaK proteins release the peptide with similar kinetics upon the addition of ATP. The DnaK K70A protein, in contrast, does not release the peptide upon the addition of ATP. ATP induces a conformational change in the wild-type, T199A, and T199S DnaK proteins but not in the DnaK K70A protein. The T199A and K70A mutations both disrupt the ATPase activity of DnaK but have profoundly different effects on the ATP-induced conformational change and peptide release activities of DnaK, implying that the two mutations affect different steps in the functional cycle of DnaK. The DnaK T199S protein represents a new class of DnaK mutant, one which has near-normal levels of ATPase activity and undergoes an ATP-induced conformational change that results in the release of peptide but which is not able to fully complement loss of DnaK function in the cell.     

Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock.

The clock gene double-time (dbt) encodes an ortholog of casein kinase Iepsilon that promotes phosphorylation and turnover of the PERIOD protein. Whereas the period (per), timeless (tim), and dClock (dClk) genes of Drosophila each contribute cycling mRNA and protein to a circadian clock, dbt RNA and DBT protein are constitutively expressed. Robust circadian changes in DBT subcellular localization are nevertheless observed in clock-containing cells of the fly head. These localization rhythms accompany formation of protein complexes that include PER, TIM, and DBT, and reflect periodic redistribution between the nucleus and the cytoplasm. Nuclear phosphorylation of PER is strongly enhanced when TIM is removed from PER/TIM/DBT complexes. The varying associations of PER, DBT and TIM appear to determine the onset and duration of nuclear PER function within the Drosophila clock.     

Expression of en and wg in the embryonic head and brain of Drosophila indicates a refolded band of seven segment remnants.

Based on the expression pattern of the segment polarity genes engrailed and wingless during the embryonic development of the larval head, we found evidence that the head of Drosophila consists of remnants of seven segments (4 pregnathal and 3 gnathal) all of which contribute cells to neuromeres in the central nervous system. Until completion of germ band retraction, the four pregnathal segment remnants and their corresponding neuromeres become arranged in an S-shape. We discuss published evidence for seven head segments and morphogenetic movements during head formation in various insects (and crustaceans).     

Further evidence that the circadian clock in Drosophila is a population of coupled ultradian oscillators

We hypothesize that ultradian oscillators are coupled to yield a composite circadian clock in Drosophila. In such a system, period would be a function of the tightness of coupling of these oscillators, increasing as coupling loosens. Ultradian oscillations would become apparent under weak coupling or in the absence of coupling. A new technique for calculating signal-to-noise ratio (SNR) for biological rhythms to characterize their precision has yielded support for this hypothesis. SNR of rhythms of the allelic series of mutations at the period (per) locus of Drosophila melanogaster were compared. Per(o) was the noisiest, grading through perL, per+, and pers, the least noisy. SNR decreases significantly with increasing period in pers, per+, and perL; per(o) typically has multiple ultradian oscillations and the lowest SNR. At least 70% of perL individuals also exhibit ultradian periodicities.     

Purification and characterization of mRNA cap-binding protein from Drosophila melanogaster embryos.

A protein with specific affinity for the mRNA cap structure was purified both from the postribosomal supernatant and from the ribosomal high-salt wash of Drosophila melanogaster embryos by m7GTP-Sepharose chromatography. This protein had an apparent molecular mass of 35 kilodaltons (kDa) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a size very different from those of the cap-binding proteins that have been characterized thus far. Drosophila 35-kDa cap-binding protein (CBP) could also be isolated from the ribosomal high-salt wash as part of a salt-stable protein complex consisting of polypeptides of 35, 72, and 140 to 180 kDa. Polyclonal antibodies against Drosophila 35-kDa CBP neither reacted with eucaryotic initiation factor 4E from rabbit reticulocytes nor affected mRNA translation in a rabbit reticulocyte cell-free system. However, in a cell-free system from Drosophila embryos, mRNA translation was specifically inhibited by these antibodies. The requirement of 35-kDa CBP for mRNA translation in Drosophila was diminished under ionic conditions in which the importance of mRNA cap structure recognition was reduced. Despite the structural differences between Drosophila 35-kDa CBP and mammalian initiation factor 4E, both proteins were functionally interchangeable in the in vitro translation system from Drosophila embryos.     

Nucleotide sequence of an mRNA transcribed in latent growth-transforming virus infection indicates that it may encode a membrane protein.

The most abundant Epstein-Barr virus mRNA in a latently infected cell line, IB4, established by in vitro growth transformation with virus, was a 2,8-kilobase RNA encoded by largely unique DNA near the right end of the genome. The RNA was transcribed from right to left, and two introns were spliced out. This region of the genome was sequenced, and the exons of the RNA were identified by S1 analysis of DNA-RNA hybrids and primer extension. The first start codon in the RNA was 40 nucleotides from its 5' end. Beginning with the start codon, there was a 1,158-nucleotide open reading frame which crossed both introns. The important characteristics of the translated protein were as follows. (i) The amino terminus was highly charged and not suggestive of a leader sequence. (ii) There were six markedly hydrophobic alpha-helical domains, each having 21 amino acids and connected by 5 to 7 amino acid segments predicted to be reverse turns. (iii) The carboxy-terminal 200 amino acids were markedly acidic, containing 6 glutamic and 37 aspartic acids. The hydrophobic region is predicted to form six membrane-spanning regions, leaving the short charged amino terminus and long acidic carboxy terminus on the inside of the membrane. This protein could be responsible for the new antigen detected in the plasma membrane of Epstein-Barr virus-transformed cells, lymphocyte-determined membrane antigen. There were two other open reading frames in the RNA.     

Analysis of the Drosophila betaPS subunit indicates that regulation of integrin activity is a primal function of the C8-C9 loop

Integrin-ligand interactions can be influenced by the sequence in a disulfide-bridged loop between the 8th and 9th beta subunit cysteines. Previous experiments are consistent with C8-C9 loop residues being involved in direct ligand-integrin interactions and/or being important in heterodimer regulation. In betaPS from Drosophila melanogaster and three other dipterans, the C8-C9 loop consists of only two amino acids, and exists in two forms that arise by differential splicing of exon 4. In these species, the betaPS4A isoform has an acidic residue in the first loop position (C8+1), with an alanine or proline in the corresponding position of betaPS4B. Mutations in both isoforms (in combination with alphaPS2) can reduce cell spreading during normal growth, but function is generally restored under conditions that enhance integrin activation. Replacement of the betaPS4A acidic residue with a basic lysine has relatively modest effects on integrin function. Spread cells bearing C8-C9 mutations tend to become less elongated, with reduced frequencies of actin stress fibers. The results indicate that even a minimal, two-residue C8-C9 loop contains structural information that can differentially regulate integrin activity and/or integrin signaling, and that this regulation does not rely on direct molecular interactions involving the variable C8+1 side chains.     

Evidence that chlorophyllin (CHLN) may behave as an inhibitor or a promoter of radiation-induced genetic damage in somatic cells of Drosophila

Irradiation of 96 h old Drosophila following a 24 h pretreatment with 5% chlorophyllin (CHLN) was delayed 0–4 days. The antimutagenic effect of CHLN in somatic cells monitored by the wing spot test persisted for 3 days after completion of the pretreatment and appeared to terminate at a time corresponding to the cessation of mitotic divisions of wing anlagen cells. Within the same population of cells, CHLN demonstrated both an inhibitory effect as measured in mwh single spot classes, and contrarily, a promoting effect in the class of mwh/flr twin spots and to an extent in the class of large flr spots. The reason for the contrasting effects of CHLN remains to be determined.     

Lack of Drosophila cytoskeletal tropomyosin affects head morphogenesis and the accumulation of oskar mRNA required for germ cell formation.

Drosophila encodes five muscle and one cytoskeletal isoform of the actin-binding protein tropomyosin. We have identified a lack-of-function mutation in the cytoskeletal isoform (cTmII). Zygotic mutant embryos show a defect in head morphogenesis, while embryos lacking maternal cTmII are defective in germ cell formation but otherwise give rise to viable adults. oskar mRNA, which is required for both germ cell formation and abdominal segmentation, fails to accumulate at the posterior pole in these embryos. nanos mRNA, however, which is required exclusively for abdominal segmentation, is localized at wild-type levels. These results indicate that head morphogenesis and the accumulation of high levels of oskar mRNA necessary for germ cell formation require tropomyosin-dependent cytoskeleton.