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PubMed2006

The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing.

BACKGROUND: Synapsins are abundant synaptic vesicle associated phosphoproteins that are involved in the fine regulation of neurotransmitter release. The Drosophila member of this protein family contains three conserved domains (A, C, and E) and is expressed in most or all synaptic terminals. Similar to mouse mutants, synapsin knock-out flies show no obvious structural defects but are disturbed in complex behaviour, notably learning and memory. RESULTS: We demonstrate that the N-terminal phosphorylation consensus motif RRxS that is conserved in all synapsins investigated so far, is modified in Drosophila by pre-mRNA editing. In mammals this motif represents the target site P1 of protein kinase A (PKA) and calcium/calmodulin dependent protein kinase I/IV. The result of this editing, by which RRFS is modified to RGFS, can be observed in cDNAs of larvae and adults and in both isolated heads and bodies. It is also seen in several newly collected wild-type strains and thus does not represent an adaptation to laboratory culture conditions. A likely editing site complementary sequence is found in a downstream intron indicating that the synapsin pre-mRNA can form a double-stranded RNA structure that is required for editing by the adenosine deaminase acting on RNA (ADAR) enzyme. A deletion in the Drosophila Adar gene generated by transposon remobilization prevents this modification, proving that the ADAR enzyme is responsible for the pre-mRNA editing described here. We also provide evidence for a likely function of synapsin editing in Drosophila. The N-terminal synapsin undeca-peptide containing the genomic motif (RRFS) represents an excellent substrate for in-vitro phosphorylation by bovine PKA while the edited peptide (RGFS) is not significantly phosphorylated. Thus pre-mRNA editing by ADAR could modulate the function of ubiquitously expressed synapsin in a cell-specific manner during development and adulthood. CONCLUSION: Similar to several other neuronal proteins of Drosophila, synapsin is modified by ADAR-mediated recoding at the pre-mRNA level. This editing likely reduces or abolishes synapsin phosphorylation by PKA. Since synapsin in Drosophila is required for various forms of behavioural plasticity, it will be fascinating to investigate the effect of this recoding on learning and memory.

Adenosine Deaminase↗
PubMed1997

PDP1, a novel Drosophila PAR domain bZIP transcription factor expressed in developing mesoderm, endoderm and ectoderm, is a transcriptional regulator of somatic muscle genes.

In vertebrates, transcriptional control of skeletal muscle genes during differentiation is regulated by enhancers that direct the combinatorial binding and/or interaction of MEF2 and the bHLH MyoD family of myogenic factors. We have shown that Drosophila MEF2 plays a role similar to its vertebrate counterpart in the regulation of the Tropomyosin I gene in the development of Drosophila somatic muscles, however, unlike vertebrates, Drosophila MEF2 interacts with a muscle activator region that does not have binding sites for myogenic bHLH-like factors or any other known Drosophila transcription factors. We describe here the isolation and characterization of a component of the muscle activator region that we have named PDP1 (PAR domain protein 1). PDP1 is a novel transcription factor that is highly homologous to the PAR subfamily of mammalian bZIP transcription factors HLF, DBP and VBP/TEF. This is the first member of the PAR subfamily of bZIP transcription factors to be identified in Drosophila. We show that PDP1 is involved in regulating expression of the Tropomyosin I gene in somatic body-wall and pharyngeal muscles by binding to DNA sequences within the muscle activator that are required for activator function. Mutations that eliminate PDP1 binding eliminate muscle activator function and severely reduce expression of a muscle activator plus MEF2 mini-enhancer. These and previous results suggest that PDP1 may function as part of a larger protein/DNA complex that interacts with MEF2 to regulate transcription of Drosophila muscle genes. Furthermore, in addition to being expressed in the mesoderm that gives rise to the somatic muscles, PDP1 is also expressed in the mesodermal fat body, the developing midgut endoderm, the hindgut and Malpighian tubules, and the epidermis and central nervous system, suggesting that PDP1 is also involved in the terminal differentiation of these tissues.

Amino Acid Sequence↗
PubMed1998

Regulation of the Tribolium homologues of caudal and hunchback in Drosophila: evidence for maternal gradient systems in a short germ embryo.

In short germ embryos, the germ rudiment forms at the posterior ventral side of the egg, while the anterior-dorsal region becomes the extra-embryonic serosa. It is difficult to see how an anterior gradient like that of bicoid in Drosophila could in these embryos be directly involved in patterning of the germ rudiment. Moreover, since it has not yet been possible to recover a bicoid homologue from any species outside the diptera, it has been speculated that the anterior bicoid gradient could be a late addition during insect evolution. We addressed this question by analysing the regulation of potential target genes of bicoid in the short germ embryo of Tribolium castaneum. We demonstrate that homologues of caudal and hunchback from Tribolium are regulated by Drosophila bicoid. In Drosophila, maternal caudal RNA is translationally repressed by bicoid. We find that Tribolium caudal RNA is also translationally repressed by bicoid, when it is transferred into Drosophila embryos under a maternal promoter. This strongly suggests that a functional bicoid homologue must exist in Tribolium. The second target gene, hunchback, is transcriptionally activated by bicoid in Drosophila. Transfer of the regulatory region of Tribolium hunchback into Drosophila also results in regulation by early maternal factors, including bicoid, but in a pattern that is more reminiscent of Tribolium hunchback expression, namely in two early blastoderm domains. Using enhancer mapping constructs and footprinting, we show that caudal activates the posterior of these domains via a specific promoter. Our experiments suggest that a major event in the evolutionary transition from short to long germ embryogenesis was the switch from activation of the hunchback gap domain by caudal to direct activation by bicoid. This regulatory switch can explain how this domain shifted from a posterior location in short germ embryos to its anterior position in long germ insects, and it also suggest how an anterior gradient can pattern the germ rudiment in short germ embryos, i.e. by regulating the expression of caudal.

Animals↗
PubMed2004

The lamin B receptor of Drosophila melanogaster.

The lamin B receptor (LBR) is an integral membrane protein of the inner nuclear membrane that has so far been characterized only in vertebrates. Here, we describe the Drosophila melanogaster protein encoded by the annotated gene CG17952 that is the putative ortholog to the vertebrate LBR. The Drosophila lamin B receptor (dLBR) has the following properties in common with the vertebrate LBR. First, structure predictions indicate that the 741 amino acid dLBR protein possesses a highly charged N-terminal domain of 307 amino acids followed by eight transmembrane segments in the C-terminal domain of the molecule. Second, immunolocalization and cell fractionation reveal that the dLBR is an integral membrane protein of the inner nuclear membrane. Third, dLBR can be shown by co-immunoprecipitations and in vitro binding assays to bind to the Drosophila B-type lamin Dm0. Fourth, the N-terminal domain of dLBR is sufficient for in vitro binding to sperm chromatin and lamin Dm0. In contrast to the human LBR, dLBR does not possess sterol C14 reductase activity when it is expressed in the Saccharomyces cerevisiae erg24 mutant, which lacks sterol C14 reductase activity. Our data raise the possibility that, during evolution, the enzymatic activity of this insect protein had been lost. To determine whether the dLBR is an essential protein, we depleted it by RNA interference in Drosophila embryos and in cultured S2 and Kc167 cells. There is no obvious effect on the nuclear architecture or viability of treated cells and embryos, whereas the depletion of Drosophila lamin Dm0 in cultured cells and embryos caused morphological alterations of nuclei, nuclear fragility and the arrest of embryonic development. We conclude that dLBR is not a limiting component of the nuclear architecture in Drosophila cells during the first 2 days of development.

Amino Acid Sequence↗
PubMed1995

Expression of Drosophila lamin C is developmentally regulated: analogies with vertebrate A-type lamins.

Vertebrate nuclear lamins form a multigene family with developmentally controlled expression. In contrast, invertebrates have long been thought to contain only a single lamin, which in Drosophila is the well-characterized lamin Dm0. Recently, however, a Drosophila cDNA clone (pG-IF) has been identified that codes for an intermediate filament protein which harbors a nuclear localization signal but lacks a carboxy-terminal CAAX motif. Based on these data the putative protein encoded by pG-IF was tentatively called Drosophila lamin C. To address whether the pG-IF encoded protein is expressed and whether it encodes a cytoplasmic intermediate filament protein or a nuclear lamin we raised antibodies against the recombinant pG-IF protein. The antibodies decorate the nuclear envelope in Drosophila Kc tissue culture cells as well as in salivary and accessory glands demonstrating that pG-IF encodes a nuclear lamin (lamin C). Antibody decoration, in situ hybridization, western and northern blotting studies show that lamin C is acquired late in embryogenesis. In contrast, lamin Dm0 is constitutively expressed. Lamin C is first detected in late stage 12 embryos in oenocytes, hindgut and posterior spiracles and subsequently also in other differentiated tissues. In third instar larvae lamins C and Dm0 are coexpressed in all tissues tested. Thus, Drosophila has two lamins: lamin Dm0, containing a CaaX motif, is expressed throughout, while lamin C, lacking a CaaX motif, is expressed only later in development. Expression of Drosophila lamin C is similar to that of vertebrate lamin A (plus C), which loses its CaaX motif during incorporation into the lamina.

Animals↗
PubMed2005

The evolution of antifungal peptides in Drosophila.

An essential component of the immune system of animals is the production of antimicrobial peptides (AMPs). In vertebrates and termites the protein sequence of some AMPs evolves rapidly under positive selection, suggesting that they may be coevolving with pathogens. However, antibacterial peptides in Drosophila tend to be highly conserved. We have inferred the selection pressures acting on Drosophila antifungal peptides (drosomycins) from both the divergence of drosomycin genes within and between five species of Drosophila and polymorphism data from Drosophila simulans and D. melanogaster. In common with Drosophila antibacterial peptides, there is no evidence of adaptive protein evolution in any of the drosomycin genes, suggesting that they do not coevolve with pathogens. It is possible that this reflects a lack of specific fungal and bacterial parasites in Drosophila populations. The polymorphism data from both species differed from neutrality at one locus, but this was not associated with changes in the protein sequence. The synonymous site diversity was greater in D. simulans than in D. melanogaster, but the diversity both upstream of the genes and at nonsynonymous sites was similar. This can be explained if both upstream and nonsynonymous mutations are slightly deleterious and are removed more effectively from D. simulans due to its larger effective population size.

Amino Acid Sequence↗
PubMed2001

A common mechanism underlies vertebrate calcium signaling and Drosophila phototransduction.

Drosophila phototransduction is an important model system for studies of inositol lipid signaling. Light excitation in Drosophila photoreceptors depends on phospholipase C, because null mutants of this enzyme do not respond to light. Surprisingly, genetic elimination of the apparently single inositol trisphosphate receptor (InsP(3)R) of Drosophila has no effect on phototransduction. This led to the proposal that Drosophila photoreceptors do not use the InsP(3) branch of phospholipase C (PLC)-mediated signaling for phototransduction, unlike most other inositol lipid-signaling systems. To examine this hypothesis we applied the membrane-permeant InsP(3)R antagonist 2-aminoethoxydiphenyl borate (2-APB), which has proved to be an important probe for assessing InsP(3)R involvement in various signaling systems. We first examined the effects of 2-APB on Xenopus oocytes. We found that 2-APB is efficient at reversibly blocking the robust InsP(3)-mediated Ca(2+) release and store-operated Ca(2+) entry in Xenopus oocytes at a stage operating after production of InsP(3) but before the opening of the surface membrane Cl(-) channels by Ca(2+). We next demonstrated that 2-APB is effective at reversibly blocking the response to light of Drosophila photoreceptors in a light-dependent manner at a concentration range similar to that effective in Xenopus oocytes and other cells. We show furthermore that 2-APB does not directly block the light-sensitive channels, indicating that it operates upstream in the activation of these channels. The results indicate an important link in the coupling mechanism of vertebrate store-operated channels and Drosophila TRP channels, which involves the InsP(3) branch of the inositol lipid-signaling pathway.

Animals↗
PubMed2003

Identification and characterization of human PRICKLE1 and PRICKLE2 genes as well as mouse Prickle1 and Prickle2 genes homologous to Drosophila tissue polarity gene prickle.

Drosophila prickle is implicated in tissue polarity or planar polarity. Here, human PRICKLE1 gene corresponding to FLJ31937 cDNA and human PRICKLE2 gene corresponding to DKFZp686D143 cDNA were identified to be homologous to Drosophila prickle gene by using bioinformatics. PRICKLE1 gene was mapped to human chromosome 12p11-q12, and PRICKLE2 gene was mapped to human chromosome 3p14. Mouse Prickle1 and Prickle2 genes were next identified in mouse genome draft sequences NW_000106.1 and NW_000262.1, respectively. Human PRICKLE1, PRICKLE2, Xenopus Prickle, and Drosophila prickle were homologous in the PET domain, three LIM domains, and the C-terminal Prickle homologous (PKH) domain. LMO6 and TESTIN, containing the PET domain and three LIM domains, were found to lack the PKH domain. Therefore, PRICKLE1 and PRICKLE2 rather than LMO6 and TESTIN were found to be human homologs of Drosophila prickle. PRICKLE1 and PRICKLE2 mRNAs were expressed together in brain, eye and testis. PRICKLE1 mRNA was expressed in fetal heart and hematological malignancies, while PRICKLE2 mRNA in fetal brain, adult cartilage, pancreatic islet, gastric cancer with signet-ring cell features, and uterus tumors. Because tissue polarity genes frizzled, dishevelled, flamingo, and Vang are evolutionary and functionary conserved from Drosophila to human, PRICKLE1 and PRICKLE2 might be implicated in the localization of Frizzled and Dishevelled proteins, just like Drosophila prickle. This is the first report on identification and characterization of human PRICKLE1, PRICKLE2, mouse Prickle1, and Prickle2 genes.

Adaptor Proteins, Signal Transducing↗
PubMed1997

Drosophila learning and memory: recent progress and new approaches.

The processes of learning and memory have traditionally been studied in large experimental organisms (Aplysia, mice, rats and humans), where well-characterized behaviors are easily tested. Although Drosophila is one of the most experimentally tractable organisms, it has only recently joined the others as a model organism for learning and memory. Drosophila behavior has been studied for over 20 years; however, most of the work in the learning and memory field has focused on initial learning, because establishing memory in Drosophila has not been as straightforward as in other organisms. A major recent advance in this field has been the development of a training protocol that induces long-term memory in files. This made possible experiments that implicated the Drosophila CREB gene as a critical component in the consolidation of long-term memory, and paves the way for future experiments utilizing the well developed tools in Drosophila. This review will briefly summarize what is known in the field of Drosophila learning and memory to date, and discuss why the unique aspects of this field make traditional approaches difficult and reward the use of alternative paths of experimentation.

Animals↗
PubMed1995

Regulation of beta-tubulin function and expression in Drosophila spermatogenesis.

In this study we examined two aspects of beta-tubulin function in Drosophila spermatogenesis: 1) beta-tubulin structural requirements for assembly of different categories of microtubules and 2) regulatory requirements for production of the correct tubulin protein level. In normal Drosophila spermatogenesis, the testis-specific beta 2-tubulin isoform supports multiple microtubule functions. Our previous work showed that another Drosophila isoform, beta 3, cannot support spermatogenesis, whereas a carboxyl-truncated form of beta 2, beta 2 delta C, can at least to some extent provide all of beta 2's normal functions, save one: beta 2 delta C cannot support organization of axonemal microtubules into the supramolecular architecture of the axoneme. Here, to test whether beta 2 carboxyl sequences can rescue the functional failure of the beta 3 isoform in spermatogenesis, we constructed a gene encoding a chimeric protein, beta 3 beta 2C, in which beta 3 sequences in the carboxyl region are replaced with those of beta 2. Unlike either beta 3 or beta 2 delta C, beta 3 beta 2C can provide partial function for both assembly of axonemal microtubules and their organization into the supramolecular architecture of the axoneme. In particular, the beta 2 carboxyl sequences mediate morphogenesis of the axoneme doublet tubule complex, including accessory microtubule assembly and attachment of spokes and linkers. However, our data also reveal aspects of beta 2-specific function that require structural features other than the primary sequence of the isotype-defining variable regions, the C terminus and the internal variable region. Tests of fecundity in males that coexpress beta 2 and the chimeric beta 3 beta 2C protein showed that in Drosophila there are differential requirements for sperm motility in the male and in the female reproductive tract. Since some aspects of microtubule function in spermatogenesis are sensitive to the tubulin pool size, we examined the mechanisms for control of tubulin protein levels in the male germ cells. We found that both beta 2-tubulin mRNA accumulation and protein synthesis are dependent on gene dose, and that the level of expression is regulated by 3' noncoding sequences in the beta 2 gene. Our data show that the regulatory mechanisms that control tubulin pool levels in the Drosophila male germ line differ from those observed in cultured animal somatic cells. Finally, expression of transgenic constructs is consistent with early cessation of X chromosome expression in Drosophila spermatogenesis.

Amino Acid Sequence↗
PubMed1982

Expression of a Drosophila heat-shock protein in Xenopus oocytes: conserved and divergent regulatory signals.

On injection of cloned Drosophila hsp70 heat-shock genes into Xenopus oocytes, heat-inducible expression is observed: the level of correctly initiated transcripts is increased 20- to 100-fold on heat shock at 34 degrees C. We show that this induction is due to activation of the heat-shock gene promoter, and that the DNA sequences required for induction lie between 10 and 66 bases upstream from the transcription start site. Most heat-induced transcripts have a correct 3' end, and hsp70 mRNA activity is detectable after extraction of the RNA from oocytes and subsequent in vitro translation. Drosophila heat-shock protein (hsp70) is synthesised in the injected oocytes after heat-shock, but only at low temperature. Under heat-shock conditions, Drosophila hsp70 mRNA translation is reduced 10-fold as is translation of the normal (25 degrees C) mRNAs, whereas translation of the endogenous Xenopus hsp70 mRNA is strongly induced. Translation of Drosophila heat-shock mRNAs extracted from flies and injected into the oocytes is also reduced by heat-shock. This suggests that Xenopus oocytes do not recognise the translational regulatory signals of Drosophila heat-shock mRNAs. In contrast, the signals for heat-induced transcription must be strongly conserved between Xenopus and Drosophila.

Animals↗
PubMed1990

Complementation of fission yeast cdc2ts and cdc25ts mutants identifies two cell cycle genes from Drosophila: a cdc2 homologue and string.

We have exploited the universality of the molecular mechanisms that control entry into mitosis to clone the Drosophila melanogaster homologues of fission yeast Schizosaccharomyces pombe cell division control (cdc) genes by the complementation of temperature sensitive mutations. The Drosophila genes were expressed in S.pombe as cDNAs from the SP6 promoter. Successful recovery of complementing plasmids required that we first 'adapt' pooled plasmids from a Drosophila embryonic cDNA library for propagation in fission yeast by introducing an ars1-LEU2 DNA fragment into the vector. This library was introduced into S.pombe cdc2 and cdc25 mutants, and plasmids isolated carrying cDNAs that complement these mutations. The gene that encodes the Drosophila cdc2 homologue maps to a single locus in the Drosophila genome at 31E on chromosome 2. It is expressed maternally to provide mRNA in syncytial embryos, and appears to be zygotically expressed in mitotically active regions of the cellularized embryo. We have isolated two different cDNAs that complement cdc25-22. One corresponds to a transcript of string, previously described as the Drosophila homologue of cdc25, and the other to a gene that has not been previously characterized.

Amino Acid Sequence↗
PubMed1993

Genetic and molecular identification of a Drosophila histidine decarboxylase gene required in photoreceptor transmitter synthesis.

Drosophila mutants of a single complementation group with defective on-/off-transients of the electroretinogram (ERG) were found to be deficient in synthesis of the photoreceptor transmitter, histamine, in a gene-dosage dependent manner, suggesting that the gene identified by the mutants (hdc) might be the structural gene for Drosophila histidine decarboxylase (HDC). A rat HDC cDNA was used to isolate a Drosophila homolog which shows approximately 60% sequence identity with mammalian HDCs over a region of 476 amino acids. In RNA blots, the Drosophila homolog detects four transcripts that are expressed primarily in the eye and are severely reduced in hdc mutants. The cloned Drosophila cDNA hybridizes to the 46F region of the chromosome, to which hdc mutations have been mapped, and rescues the hdc mutant phenotype in transgenic flies generated by P element-mediated germline transformation. The results thus show that the Drosophila homolog corresponds to the histidine decarboxylase gene, identified by the hdc mutants, and that mutations in the gene disrupt photoreceptor synaptic transmission.

Amino Acid Sequence↗
PubMed2003

Targeted gene expression in Drosophila dopaminergic cells using regulatory sequences from tyrosine hydroxylase.

Dopamine (DA) is the only catecholaminergic neurotransmitter in the fruit fly Drosophila melanogaster. Dopaminergic neurons have been identified in the larval and adult central nervous system (CNS) in Drosophila and other insects, but no specific genetic tool was available to study their development, function, and degeneration in vivo. In Drosophila as in vertebrates, the rate-limiting step in DA biosynthesis is catalyzed by the enzyme tyrosine hydroxylase (TH). The Drosophila TH gene (DTH) is specifically expressed in all dopaminergic cells and the corresponding mutant, pale (ple), is embryonic lethal. We have performed ple rescue experiments with modified DTH transgenes. Our results indicate that partially redundant regulatory elements located in DTH introns are required for proper expression of this gene in the CNS. Based on this study, we generated a GAL4 driver transgene, TH-GAL4, containing regulatory sequences from the DTH 5' flanking and downstream coding regions. TH-GAL4 specifically expresses in dopaminergic cells in embryos, larval CNS, and adult brain when introduced into the Drosophila genome. As a first application of this driver, we observed that in vivo inhibition of DA release induces a striking hyperexcitability behavior in adult flies. We propose that TH-GAL4 will be useful for studies of the role of DA in behavior and disease models in Drosophila.

Animals↗
PubMed1986

Developmental effects of chemicals and the heat shock response in Drosophila cells.

Exposure of prokaryotic and eukaryotic cells to heat shock (hyperthermia) or to a number of diverse environmental stresses such as teratogens, anoxia, and inhibitors of oxidative phosphorylation results in the enhanced synthesis of a number of proteins which have been previously referred to as heat shock proteins (hsps). More recently, in view of the diverse types of agents that can induce these proteins, they have also been referred to as stress proteins. This phenomenon is one of the most basic regulatory mechanisms in living organisms. Exposure of Drosophila embryos, larvae, or pupae to these types of stresses also results in a variety of developmental abnormalities in the ensuing adult. Although the function(s) of these heat shock proteins has yet to be determined, they are widely thought to play an important role in cell survival and protection following some types of environmental stress. In our laboratory, we have developed an in vitro assay for detecting agents that act as teratogens, utilizing Drosophila embryonic cultures. Drosophila embryonic cells differentiate in vitro to a number of functional cell types including myotubes and ganglia. A number of drugs that have been shown to act as teratogens in mammals have also been found to inhibit muscle and/or neuron differentiation in Drosophila embryonic cultures. We have examined, by two-dimensional gel electrophoresis, the effects of such teratogens on protein synthesis in Drosophila embryonic cells. Inhibition of muscle and/or neuron differentiation correlates well with the induction of two proteins of about 20 kilodaltons. These are identical to two of the heat shock proteins (hsp 23, 22) as shown by electrophoretic mobilities and peptide mapping by partial proteolysis. Heat shock and other treatments such as exposure to some of the metal ions and ether induces the entire set of seven major heat shock proteins in the Drosophila embryonic cells. Dose-response studies of several teratogens show a correlation between the degree of inhibition of differentiation and the level of induction of hsps. Since heat shock proteins have been suggested as possibly serving a protecting role, our present studies are aimed at identifying the role of hsps in teratogenesis and investigating the differential regulation of heat shock genes in response to different external stimuli.

Animals↗
PubMed2000

Molecular cloning and genomic organization of a novel receptor from Drosophila melanogaster structurally related to mammalian galanin receptors.

We screened the Berkeley "Drosophila Genome Project" database with "electronic probes" corresponding to conserved amino acid sequences from the five known rat somatostatin receptors. This yielded alignment with a Drosophila genomic clone that contained a DNA sequence coding for a protein, having amino acid sequence identities with the rat galanin receptors. Using PCR with Drosophila cDNA as a template, and oligonucleotide probes coding for the exons of the presumed Drosophila gene, we were able to clone the cDNA for this receptor. The Drosophila receptor has most amino acid sequence identity with the three mammalian galanin receptors (37% identity with the rat galanin receptor type-1, 32% identity with type-2, and 29% identity with type-3). Less sequence identity exists with the mammalian opioid/nociceptin-orphanin FQ receptors (26% identity with the rat micro opioid receptor), and mammalian somatostatin receptors (25% identity with the rat somatostatin receptor type-2). The novel Drosophila receptor gene contains ten introns and eleven exons and is located at the distal end of the X chromosome.

Amino Acid Sequence↗
PubMed1995

Localization of sequences required for size-specific splicing of a small Drosophila intron in vitro.

Many introns in Drosophila and other invertebrates are less than 80 nucleotides in length, too small to be recognized by the vertebrate splicing machinery. Comparison of nuclear splicing extracts from human HeLa and Drosophila Kc cells has revealed species-specificity, consistent with the observed size differences. Here we present additional results with the 68 nucleotide fifth intron of the Drosophila myosin heavy chain gene. As observed with the 74 nucleotide second intron of the Drosophila white gene, the wild-type myosin intron is accurately spliced in a homologous extract, and increasing the size by 16 nucleotides both eliminates splicing in the Drosophila extract and allows accurate splicing in the human extract. In contrast to previous results, however, an upstream cryptic 5' splice site is activated when the wild-type myosin intron is tested in a human HeLa cell nuclear extract, resulting in the removal of a 98 nucleotide intron. The size dependence of splicing in Drosophila extracts is also intron-specific; we noted that a naturally larger (150 nucleotide) intron from the ftz gene is efficiently spliced in Kc cell extracts that do not splice enlarged introns (of 84, 90, 150 or 350 nucleotides) derived from the 74 nucleotide white intron. Here, we have exploited that observation, using a series of hybrid introns to show that a region of 46 nucleotides at the 3' end of the white intron is sufficient to confer the species-specific size effect. At least two sequence elements within this region, yet distinct from previously described branchpoint and pyrimidine tract signals, are required for efficient splicing of small hybrid introns in vitro.

Animals↗
PubMed1999

Drosophila replication and repair proteins: proliferating cell nuclear antigen (PCNA).

Proliferating cell nuclear antigen (PCNA), a protein intimately involved in both replication and repair, has been identified in eukaryotes at all levels of evolution. Is primary sequence, Drosophila melanogaster PCNA is 73% identical to mammalian PCNA. Moreover, it is able to substitute for mammalian PCNA in at least one intricate cell-free replication assay. Mutations in the gene for Drosophila PCNA, including some that are temperature sensitive, have been reported. Procedures are described for the biochemical purification of wild-type PCNA from a population of 6- to 18-h-old Drosophila embryos. Procedures were also developed for purification of unmodified wild-type Drosophila PCNA after induction of expression in Escherichia coli. An NH(2)-terminally His-tagged but otherwise wild-type Drosophila PCNA, as well as mutant His-tagged PCNA, were also engineered and purified to apparent homogeneity. Finally, an in situ polyacrylamide gel technique allows DNA polymerase assays to be performed on portions of single adults as well as single Drosophila embryos. This assay should tremendously facilitate systematic genetic studies of metazoan replication and repair.

Animals↗