Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles.

The microtubule-binding protein tau has been implicated in the pathogenesis of Alzheimer's disease and related disorders. However, the mechanisms underlying tau-mediated neurotoxicity remain unclear. We created a genetic model of tau-related neurodegenerative disease by expressing wild-type and mutant forms of human tau in the fruit fly Drosophila melanogaster. Transgenic flies showed key features of the human disorders: adult onset, progressive neurodegeneration, early death, enhanced toxicity of mutant tau, accumulation of abnormal tau, and relative anatomic selectivity. However, neurodegeneration occurred without the neurofibrillary tangle formation that is seen in human disease and some rodent tauopathy models. This fly model may allow a genetic analysis of the cellular mechanisms underlying tau neurotoxicity.

125I-Labeled alpha-bungarotoxin and [3H]quinuclidinylbenzilate binding to rat brain membranes. Effects of physical, chemical and enzymatic treatments.

The effects of various physical, chemical and enzymatic treatments of rat brain membranes were investigated with respect to 125I-labeled alpha-bungarotoxin ([125I]alpha BuTx) and [3H]quinuclidinylbenzilate ([3H]QNB) binding. Binding appeared relatively stable to autolysis, mechanical shearing, freeze-thawing, and divalent cation addition (Sr2+) or removal (EGTA, EDTA). Binding for [125I]-alpha BuTx was slightly reduced by trypsin digestion of the membranes while both [125I]alpha BuTx and [3H]QNB binding were reduced by phospholipase A2 digestion (Crotalus adamantus phospholipase A2 and beta-bungarotoxin). Treatment of the membranes with the disulfide reducing agent, dithiothreitol, resulted in additional [125I]alpha BuTx binding but showed little effect on [3H]QNB binding. Binding of the cholinergic agonists, nicotine and carbamylcholine, was studied by observing their concentration-dependent ability to inhibit [125I]alpha BuTx and [3H]QNB binding, respectively. Membrane sulfhydryl group reduction and endogenous cation removal by EGTA or EDTA resulted in a lowered affinity for nicotine bindng. Alkylation of membranes with N-ethylmaleimide resulted in an increase in carbamylcholine affinity. Other treatments had little or no effect on nicotine or carbamylcholine binding.

Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.

The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.

Molecular biology and neurobiology of choline acetyltransferase.

In the 45 years since the first description of choline acetyltransferase (ChAT; EC 2.3.1.6.), significant progress has been made in characterizing the molecular properties of this important neurotransmitter synthetic enzyme. We are now on the verge of understanding its genetic regulation and biological function(s). The Drosophila cDNA has been cloned, sequenced, and expressed in both a eucaryotic and a procaryotic system. The levels of ChAT specific mRNA have been determined during Drosophila development. Monoclonal and polyclonal antibodies have been produced to the enzyme from a variety of sources and used for biochemical and immunocytochemical studies. Two well characterized genetic systems have identified the ChAT gene and described a series of useful alleles. As a nervous system specific protein expressed only in the subset of neurons using acetylcholine as a neurotransmitter, ChAT is a good model for uncovering the processes and factors responsible for regulating genes involved in neurotransmitter phenotype selection and maintenance. Recent studies have described the purification of a cholinergic factor from muscle conditioned medium and indicated the potential importance of nerve growth factor (NGF) for regulating ChAT expression in the central nervous system. These factors, or ones remaining to be discovered, may be involved in the etiology or disease process of neurodegenerative nervous system disorders such as Alzheimer's disease.

Organization and transcriptional regulation of Drosophila Na(+), K(+)-ATPase beta subunit genes: Nrv1 and Nrv2.

Drosophila melanogaster has two Na(+),K(+)-ATPase beta subunit genes (Nervana 1 and 2; Nrv), with tissue-specific expression patterns. Nrv1 produces a single beta subunit isoform expressed primarily in muscle tissue, whereas Nrv2 codes for two different isoforms (2.1 and 2.2) expressed in the nervous system. We have determined the complete molecular genomic organization for both Nrv genes. Only 3kb of DNA separate the 3' end of Nrv2 from Nrv1. The cDNAs from all three forms of Nrv have been mapped onto the genomic structure and all intron-exon junctions have been confirmed by direct sequencing. The genomic DNA positioned in the 5' flanking region of each Nrv gene has also been tested for tissue-specific transcriptional regulatory activity. P-element transformation vectors were constructed, which contained either 7.7kb of Nrv2 or 3.5kb Nrv1 5' flanking DNA driving expression of a lacZ reporter gene. Multiple transgenic Drosophila lines were established for each construct and analyzed for their beta-galactosidase expression pattern. The tissue-specific expression of each Nrv gene is independently regulated by the cis-element(s) present in the 5' flanking region. The Nrv2 5' flanking DNA directs expression exclusively to the nervous system, whereas Nrv1 5' flanking DNA directs expression primarily in muscle tissue.

Immunocytochemical study of a temperature-sensitive choline acetyltransferase mutant of Drosophila melanogaster.

Using a monoclonal antibody to choline acetyltransferase (ChAT), we have identified immunoreactive synaptic terminals in the neuropil regions of the cephalic ganglion of Drosophila melanogaster. This study demonstrates the distribution of antibody-labeled structures within the optic lobe, and then investigates the immunoreactivity altered by mutation in two temperature-sensitive ChAT alleles, chats-1 and chats-2. The general structure of the optic lobe was first observed by means of the silver impregnation technique. Then the presence of ChAT immunoreactivity was determined by the application of antibody [1G4] conjugated with HRP to frozen sections, followed by the 3,3'-diamino-benzidine tetratinct layers, which correspond to the three synaptic layers of the laminarneurons, in the medulla. Also, staining appeared in four distinct layers in the lobula. In addition, weaker staining was observed in the lamina, which corresponds to the retinula cell terminals. Somal layers were not stained. In Canton-S (wild-type), the three medullar layers stain distinctly at both 19 degrees C and 30 degrees C. In chats-1 at 19 degrees C, the stain appeared in the same layers as that of Canton-S, but with somewhat lower density. In chats-2 at 19 degrees C, the density of the stain was even lower. The densities of the stain in these mutants were further decreased after exposing the flies to 30 degrees C. The decreases were dependent on the length of exposure to the higher temperature. The decrease in stain of the specimens obtained after 24 hours exposure to 30 degrees C was clearly recognizable in both chats-1 and chats-2.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunocytochemical study of choline acetyltransferase in Drosophila melanogaster: an analysis of cis-regulatory regions controlling expression in the brain of cDNA-transformed flies.

We have analyzed the cis-regulatory regions in the 5' flanking DNA of the Drosophila melanogaster choline acetyltransferase (ChAT; E.C. 2.3.1.6) gene by using germline transformants. These transformants are carrying wild-type ChAT cDNA fused to different lengths of 5' flanking sequence of the ChAT gene. Appropriate genetic crosses were used to introduce the transgene into animals with a presumptive null genetic background for endogenous ChAT. Expression of ChAT protein could thus be attributed exclusively to the transgene. Using a monoclonal antibody against Drosophila ChAT, we have investigated the spatial distribution of transgenic ChAT and compared it to the normal distribution of ChAT protein in wild-type animals. The brains of 7.4 kb cDNA transformants showed a ChAT expression pattern similar to that of wild-type animals in the first- and second-order sensory neuropil but reduced expression in other highly ordered neuropil. Several lines that were transformed with 1.2 kb or 0.8 kb of 5' flanking DNA demonstrated relatively normal expression in sensory neuropil. In addition, these lines also showed ectopic expression in higher order neuropil. In the optic lobe, the expression pattern directed by 7.4 kb of 5' flanking DNA was very similar to that of wild-type ChAT expression. In contrast, 1.2 kb or 0.8 kb transformants showed reduced levels of expression and a more limited pattern of distribution in the optic lobe. Our results suggest that the 5' flanking DNA of the ChAT gene can be divided into several separable positive and negative regulatory regions, which define various subsets of cholinergic neurons in the nervous system.

Immunocytochemical localization of choline acetyltransferase in rat cerebral cortex: a study of cholinergic neurons and synapses.

Choline acetyltransferase (ChAT), the acetylcholine-synthesizing enzyme and a definitive marker for cholinergic neurons, was localized immunocytochemically in the motor and somatic sensory regions of rat cerebral cortex with monoclonal antibodies. ChAT-positive (ChAT+) varicose fibers and terminal-like structures were distributed in a loose network throughout the cortex. Some immunoreactive cortical fibers were continuous with those in the white matter underlying the cortex, and many of these fibers presumably originated from subcortical cholinergic neurons. ChAT+ fibers appeared to be rather evenly distributed throughout all layers of the motor cortex, but a subtle laminar pattern was evident in the somatic sensory cortex, where lower concentrations of fibers in layer IV contrasted with higher concentrations in layer V. Electron microscopy demonstrated that immunoreaction product was concentrated in synaptic vesicle-filled profiles and that many of these structures formed synaptic contacts. ChAT+ synapses were present in all cortical layers, and the majority were of the symmetric type, although a few asymmetric ones were also observed. The most common postsynaptic elements were small to medium-sized dendritic shafts of unidentified origin. In addition, ChAT+ terminals formed synaptic contacts with apical and, probably, basilar dendrites of pyramidal neurons, as well as with the somata of ChAT-negative nonpyramidal neurons. ChAT+ cell bodies were present throughout cortical layers II-VI, but were most concentrated in layers II-III. The somata were small in size, and the majority of ChAT+ neurons were bipolar in form, displaying vertically oriented dendrites that often extended across several cortical layers. Electron microscopy confirmed the presence of immunoreaction product within the cytoplasm of small neurons and revealed that they received both symmetric and asymmetric synapses on their somata and proximal dendrites. These observations support an identification of ChAT+ cells as nonpyramidal intrinsic neurons and thus indicate that there is an intrinsic source of cholinergic innervation of the rat cerebral cortex, as well as the previously described extrinsic sources.

Ultrastructural characterization of identified cholinergic neurons transplanted to the hippocampal formation of the rat.

The ultrastructural features of cholinergic neurons transplanted to the rat hippocampal formation were studied by using a monoclonal antibody to choline acetyltransferase (ChAT). Septal cell suspensions were prepared from E-18 rat embryos and injected into the hippocampus of host rats that had been previously subjected to a bilateral transection of the fimbria-fornix. Rats with fimbria-fornix lesions alone and unoperated rats served as controls and were examined to characterize the native hippocampal cholinergic system. Both unoperated controls and rats with fimbria-fornix lesions showed a sparse population of intrinsic ChAT-immunoreactive neurons that were most numerous in the subgranular zone, the hilus fascia dentata, and near the hippocampal fissure. ChAT-positive terminals from controls formed synapses on dendritic structures that were primarily symmetrical. ChAT-positive dendrites in controls received synaptic input from nonimmunoreactive axon terminals. In rats with septal transplants, ChAT-immunoreactive transplant neurons were found that were either bipolar or multipolar. Axons of transplanted neurons were unmyelinated and arose either from the cell body or a primary dendritic process where they gave off numerous collaterals. Terminals from transplant neurons formed synapses with many nonimmunoreactive neurons. In transplant animals, two main targets of ChAT-immunoreactive terminals were identified: The great majority of synapses were symmetrical junctions with dendritic spines and shafts. A number of terminals were found that appeared to be juxtaposed to nonimmunoreactive axon terminals, possibly forming symmetrical axo-axonic connections. In contrast, such axo-axonic contacts were not observed in the controls. It is concluded that transplanted cholinergic neurons may reinnervate the host hippocampus; however, this reinnervation is different from what is seen in the intact hippocampal formation.

Localization of Drosophila neurons that contain choline acetyltransferase messenger RNA: an in situ hybridization study.

In situ hybridization with radiolabeled complementary RNA (cRNA) probes was used to determine the location of the messenger RNA (mRNA) encoding choline acetyltransferase (ChAT) in Drosophila nervous system. Areas in the cell-rich cortical regions of the cerebrum and optic lobes hybridized with substantial concentrations of the probe. This contrasted with the cell-sparse neuropil areas where no significant concentrations of probe were observed. Although most of the cortical regions were substantially labeled, there were regions within all of the areas where labeling was sparse or nonexistent. For example in the lamina, even though the monopolar cell layer appeared to be heavily labeled, there were some neuronal profiles that were not associated with the probe. Moreover, the epithelial glia that form an arch of cell profiles subjacent to the monopolar cells were not labeled, nor were amacrine neurons in the apex of the lamina near the external optic chiasma. The highest concentration of probe (approximately 140 grains/400 microns2) was observed in the laminar monopolar cell region and the cerebral cortical rind. The next most heavily labeled region (approximately 90 grains/400 microns2) occurred over cortical cells of the medulla-lobula. In the peripheral nervous system, label over the antennal sensory neurons amounted to about 75 grains/400 microns2, and the retinular cell layer of the compound eye exhibited about 60 grains/400 microns2. The control probe did not hybridize in significant quantities in either cellular or noncellular regions. This study presents evidence that large numbers of Drosophila cortical and primary sensory neurons contain the messenger RNA necessary for the production of ChAT, the acetylcholine-synthesizing enzyme. Further, our findings provide baseline information for use in ontogenetic studies of cholinergic neurons in Drosophila, and they also provide normative data for studying the effects of mutant alleles at the Cha or Ace loci upon the transcription of ChAT messenger RNA.

Embryonic development of four different subsets of cholinergic neurons in rat cervical spinal cord.

The developmental stage at which a neuron becomes committed to a neurotransmitter phenotype is an important time in its ontogenetic history. The present study examines when choline acetyltransferase (ChAT) is first detected within each of four different subsets of cholinergic neurons previously identified in the cervical enlargement of the spinal cord: namely, motor neurons, partition cells, central canal cluster cells, and dorsal horn neurons. By examining the temporal sequence of embryonic development of these cholinergic neurons, we can infer the relationships between ChAT expression and other important developmental events. ChAT was first detected reliably on embryonic day 13 (E13) by both biochemical and immunocytochemical methods, and it was localized predominantly within motor neurons. A second group of primitive-appearing ChAT-positive cells was detected adjacent to the ventricular zone on E14. These neurons seemed to disperse laterally into the intermediate zone by E15, and, on the basis of their location, were tentatively identified as partition cells. A third group of primitive ChAT-immunoreactive cells was detected on E16, both within and around the ventral half of the ventricular zone. By E17, some members of this "U"-shaped group appeared to have dispersed dorsally and laterally, probably giving rise to dorsal horn neurons as well as dorsal central canal cluster cells. Other members of this group remained near the ventral ventricular zone, most likely differentiating into ventral central canal cluster cells. Combined findings from the present study and a previous investigation of neurogenesis (Phelps et al.: J. Comp. Neurol. 273:459-472, '88), suggest that premitotic precursor cells have not yet acquired the cholinergic phenotype because ChAT is not detectable until after the onset of neuronal generation for each of the respective subsets of cholinergic neurons. However, ChAT is expressed in primitive bipolar neurons located within or adjacent to the germinal epithelium. Transitional stages of embryonic development suggest that these primitive ChAT-positive cells migrate to different locations within the intermediate zone to differentiate into the various subsets of mature cholinergic neurons. Therefore, it seems likely that spinal cholinergic neurons are committed to the cholinergic phenotype at pre- or early migratory stages of their development. Our results also hint that the subsets of cholinergic cells may follow different migration routes. For example, presumptive partition cells may use radial glial processes for guidance, whereas dorsal horn neurons may migrate along nerve fibers of the commissural pathway. Cell-cell interactions along such diverse migratory pathways could play a role in determining the different morphological, and presumably functional, phenotypes expressed by spinal cholinergic neurons.

The morphology and distribution of neurons containing choline acetyltransferase in the adult rat spinal cord: an immunocytochemical study.

A monoclonal antibody to choline acetyltransferase (ChAT), the acetylcholine (ACh)-synthesizing enzyme, has been used to localize ChAT within neurons in immunocytochemical preparations of adult rat spinal cord. Morphological details of known cholinergic spinal neurons are presented in this study, and previously unidentified ChAT-containing neurons are also described. Immunoreaction product was present within cell bodies, dendrites, axons, and axon terminals, thereby allowing comprehensive descriptions of the distribution of ChAT-positive neurons and the interrelationships of their processes. In the ventral horn, ChAT-positive motoneurons were located in the medial, central, and lateral motor columns, and their dendrites formed elaborate longitudinal and transverse ChAT-positive bundles. These bundles were present throughout the rostrocaudal extent of the spinal cord. In the central gray matter, small ChAT-positive cell bodies were clustered around the central canal. Small longitudinal fascicles of immunoreactive processes were also observed in this region adjacent to the ependymal layer. The intermediate gray matter of virtually the entire spinal cord was spanned by medium to large ChAT-positive multipolar cells termed partition neurons. At autonomic spinal levels, partition neurons were intermingled with other immunoreactive cells that were identified as preganglionic sympathetic or parasympathetic neurons because of their locations and morphological characteristics. In the sympathetic system, four groups of ChAT-positive neurons were observed; the principal intermediolateral nucleus (ILp) in the lateral horn, the central autonomic cell column (CA) dorsal to the central canal, the intercalated nucleus (IC) located between ILp and CA, and the funicular intermediolateral neurons (ILf) in the white matter lateral to the ILp. The dendrites of ILp and CA neurons formed substantial longitudinal bundles within each group, as well as transverse bundles between the groups that resembled the rungs of a ladder. ChAT-positive cell bodies were also present in the dorsal horn, and those located in laminae III-V extended dendrites dorsally into a longitudinal plexus within lamina III.

Postnatal development of neurons containing choline acetyltransferase in rat spinal cord: an immunocytochemical study.

A monoclonal antibody to choline acetyltransferase (ChAT) has been used in an immunocytochemical study of the postnatal development of ChAT-containing neurons in cervical and thoracic spinal cord. Specimens from rat pups ranging in age from 1 to 28 days postnatal (dpn) were studied and compared with adult specimens (Barber et al., '84). The development of established cholinergic neurons, the somatic motoneurons and sympathetic preganglionic cells, has been described as has that of previously unidentified ChAT-positive neurons in the dorsal, intermediate, and central gray matter. Cell bodies of somatic and visceral motoneurons contained moderate amounts of ChAT-positive reaction product at birth that gradually increased in intensity until 14-21 dpn. The most intensely stained ChAT-positive neurons in 1-5-dpn specimens were named partition cells because this cell group extended from the central gray to an area dorsal to the lateral motoneurons, and thereby divided the spinal cord into dorsal and ventral halves. Partition cells were medium to large in size with 5-7 primary dendrites, and axons that, in fortuitous sections, could be traced into the ventrolateral motoneuron pools, the ventral funiculi, or the ventral commissure. Small ChAT-positive cells clustered around the central canal and scattered in laminae III-VI of the dorsal horn were detectable at birth. These neurons were moderately immunoreactive at 11-14 dpn and intensely ChAT positive by 21 dpn. The band of ChAT-positive terminal-like structures demonstrated in lamina III of adult specimens (Barber et al., '84) was first visible in 11-14-dpn specimens. By 28 dpn, both laminae I and III contained punctate bands that approximated the density of those observed in adult spinal cord. This investigation has demonstrated ChAT within individual neurons of developing spinal cord, and has identified a group of neurons, the partition cells, that exhibit intense ChAT-positive immunoreactivity earlier than any other putative cholinergic cells in spinal cord, including motoneurons. Another important observation has been that each ChAT-positive neuronal type achieves adult levels of staining intensity at different times during development. A likely explanation for this differential staining is that various groups of neurons acquire their mature concentration of ChAT molecules at different developmental stages. In turn, this may correlate with the maturation of cholinergic synaptic activity manifest by individual cells or groups of neurons.

Sequence of choline acetyltransferase temperature-sensitive mutants determined by the polymerase chain reaction.

Two temperature-sensitive alleles of Drosophila melanogaster, Cha(ts1) and Cha(ts2), have been previously identified and are thought to be point mutations in the structural gene for the neurotransmitter biosynthetic enzyme choline acetyltransferase. In order to clarify the molecular nature of these alleles and characterize the presumed amino acid substitutions, we have used the polymerase chain reaction to amplify choline acetyltransferase messenger ribonucleic acid fragments from both mutant genotypes. Amplified mutant complementary deoxyribonucleic acid was cloned and used to construct chimeric complementary deoxyribonucleic acid clones containing approximately two-thirds of the wild-type sequence which would code for N-terminal amino acids and one-third of the mutant sequence coding for the C-terminal amino acids. After in vitro translation of complementary ribonucleic acid produced from the chimeric complementary deoxyribonucleic acid clones, choline acetyltransferase activity was determined and shown to be thermolabile. Sequence analyses of these clones showed that one amino acid substitution due to single base substitution is crucial in each chimeric choline acetyltransferase complementary deoxyribonucleic acid to generate a thermolabile choline acetyltransferase product. The point mutations of the structural gene for choline acetyltransferase are thus confirmed and shown to regulate the thermolability of the enzyme produced by Cha(ts1) and Cha(ts2).

Preparation and characterization of peroxidase: antiperoxidase-Fab complex.

The preparation and characterization of a horseradish peroxidase-rabbit antiperoxidase Fab immunocomplex (HRP-Fab2) useful for immunocytochemical localization of primary tissue-bound rabbit antibody are described. Antisera with titer to horseradish peroxidase (HRP) were raised in rabbits. Anti-HRP-Fab fragments were prepared by controlled mercuripapain digestion of the purified rabbit IgG. The complex was formed during incubation of Fab fragments with HRP, and fractions containing HRP activity that were precipitable by goat anti-rabbit IgG serum were isolated by gel filtration. The major isolated complex had a molecular weight of approximately 150,000 daltons and migrated as a single band on cellulose acetate electrophoresis. Polyacrylamide gel electrophoresis in SDS indicated the major polypeptide components of the complex were HRP and Fab. RZ (absorbance at 403 nm/275 nm) determination indicated a molar ratio of 2 Fab:1 HRP. The complex was stable for at least 1 year at -20 degrees C and was used successfully in a number of immunocytochemical procedures.

Species-specific second antibodies reduce spurious staining in immunocytochemistry.

Spurious staining related to the second (linking) antibodies was observed in immunocytochemical specimens processed with an unlabeled antibody method. Some of this staining was suspected to result from species cross-reactivity of the second antibodies with endogenous immunoglobulin Gs in the tissue. Therefore, species-specific second antibodies were obtained, and the staining patterns of tissue processed with such antibodies were compared with those of tissue processed with standard (nonspecies-specific) second antibodies. In these studies, a monoclonal antibody to choline acetyltransferase (ChAT) was utilized as the primary antibody, and a similarly prepared monoclonal antibody that did not react with ChAT served as a control antibody. Spurious staining that included staining of discrete tissue and cellular components as well as amorphous background staining was present in both control and experimental tissue processed with standard second antibodies. Such staining was virtually eliminated in tissue processed with species-specific second antibodies. In specimens from the central nervous system, for example, species-specific second antibodies greatly reduced dark staining within the area postrema, in the pia-arachnoid membranes, and around blood vessels as well as the staining of small dot-like structures within some large neurons. In addition, the general level of background staining was reduced in both adult and developing tissues, thus permitting clearer visualization of many positively stained structures. In peripheral tissues such as skeletal muscle, spurious staining of connective tissue elements was eliminated, allowing the observation of previously occluded ChAT-positive structures such as nerve fibers and motor end-plates. Thus, species-specific second antibodies appear to be very useful for immunocytochemistry, particularly when the primary antibody and the tissue to be studied are from closely related species.

Biochemical analysis of proteins recognized by anti-HRP antibodies in Drosophila melanogaster: identification and characterization of neuron specific and male specific glycoproteins.

Antibodies recognizing horse radish peroxidase (HRP) stain neurons in Drosophila and other insects. We have used Western blots to analyze and characterize some of the anti-HRP reactive components from Drosophila melanogaster. Anti-HRP reactive components can be reproducibly detected during all developmental stages, although the pattern changes at different developmental times. In adults, there are at least 10 reproducibly stained components. Two of the bands, with molecular sizes of 42 and 80 kDa are likely to be the major contributors to neuronal anti-HRP staining in Drosophila. These components are enriched in adult fly heads. In contrast, many of the other anti-HRP reactive components in adults are enriched in abdomen and are present exclusively or at much higher levels in male flies. We have purified and characterized two of the male specific components with molecular sizes of 62 and 48 kDa. Partial N-terminal amino acid sequencing revealed that the 62 kDa protein is identical to a part of D. melanogaster carboxylesterase to EC 3.1.1.1) while he 48 kDa protein does not match any known sequences. Esterase-6 has previously been shown to be enriched in male accessory gland and consistent with this we show staining of this structure with anti-HRP antibodies.

Positive and negative feedback regulation of choline acetyltransferase mRNA levels in Drosophila: a study using temperature-sensitive mutants and embryo cell cultures.

Steady state levels of the mRNA coding for the neurotransmitter biosynthetic enzyme, acetylCoA-choline-O-acetyltransferase (ChAT, EC 2.3.1.6), were measured in wild type Drosophila and two temperature-sensitive mutants (Chats1 and Chats2) using the RNase protection method. At a permissive temperature the relative amounts of ChAT mRNA were: wild type: Chats1:Chats2 = 1:2.09 (+/- 0.39):3.37 (+/- 0.57) (mean +/- S.E.M.) indicating that mutant flies may compensate, for making a thermolabile form of enzyme, by producing and/or maintaining higher levels of ChAT mRNA. At a restrictive temperature the ChAT mRNA levels decreased in both mutants and increased in wild type flies. The regulatory mechanism(s) responsible for increasing ChAT mRNA in wild type flies appears to have failed in the mutants at high temperature. Steady state mRNA levels were also measured in embryonic cell cultures prepared from wild type embryos. Cultures grown in the presence of two pharmacologic agents (carbamylcholine and d-tubocurarine) which should interfere with cholinergic neurotransmission, showed less mRNA resulting from a decrease in levels of ChAT gene transcription. Our results imply that neurotransmission and the rate of neurotransmitter biosynthetic enzyme gene transcription are coupled for the cholinergic system in Drosophila.

Partial cloning of the rat choline acetyltransferase gene and in situ localization of its transcripts in the cell body of cholinergic neurons in the brain stem and spinal cord.

We have isolated recombinant lambda (lambda) phages which contain a part of the rat choline acetyltransferase (ChAT) gene. Restriction and Southern blot analyses using synthetic oligonucleotides indicate that these clones overlap one another and contain at least four exons which reside in 16.4 kb of sequence encoding from the middle to the 3' end, but not the 5'-region, of the rat ChAT gene. Partial sequence analyses revealed that the clones contain an exon whose nucleotide sequence corresponds to a highly conserved region of ChAT during evolution. RNase protection mapping experiments show that sequences represented by this exon are expressed at high levels in the spinal cord of adult rats and at low but detectable levels in PC12 cells. By using the genomic sequences, including the exon, as a hybridization probe, we have detected ChAT mRNAs in situ in rat tissues. In situ hybridization experiments using radioactive and non-radioactive probes revealed that cholinergic motoneurons in the spinal cord, the laterodorsal tegmental nucleus as well as the hypoglossal nucleus in the brain stem were labeled, suggesting that the genomic sequence can be used as a probe to measure the ChAT mRNA levels in those cholinergic neurons. The results also indicate that the non-radioactive method gives a better resolution in localizing the expression of ChAT transcripts in the cytoplasm of cholinergic neurons.

Molecular characterization of choline acetyltransferase from Drosophila melanogaster.

Purified acetyl-CoA: choline O-acetyltransferase (EC 2.3.1.6) from Drosophila melanogaster has been shown to contain two major polypeptides of 67 and 54 K Daltons. However, all enzyme activity is found in a single molecular weight form of approx 67 K Daltons as determined by sucrose gradient sedimentation and molecular exclusion chromatography. The latter showed both the 67 and 54 K Dalton polypeptides on polyacrylamide gel electrophoresis in sodium lauryl sulfate (10% acrylamide). Analysis of purified choline acetyltransferase on polyacrylamide gel electrophoresis in sodium lauryl sulfate (15% acrylamide) revealed the presence of an additional polypeptide at 13 K Daltons. Tryptic-peptide maps of the 67, 54 and 13 K Dalton components showed all three to be structurally related. In addition to several common tryptic peptides, the 13 K Dalton polypeptide contained three tryptic-peptides that were also found in the 67 K Dalton polypeptide, but were absent from the 54 K Dalton polypeptide. This evidence suggests that native Drosophila choline acetyltransferase may exist in two forms, one a single polypeptide chain with a molecular weight of 67 K Daltons and the other consisting of two noncovalently bound polypeptide chains with molecular weights of 54 and 13 K Daltons. The latter form is the major one isolated and may be generated by limited proteolysis of the single chain 67 K Dalton form.