Coinjection of Xenopus oocytes with cDNA-produced and native mRNAs: a molecular biological approach to the tissue-specific processing of human cholinesterases.
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Acetylcholinesterase↗
Biomedical subjects
H Soreq
Publications and source records attributed to H Soreq.
Expression and in vivo amplification of the human acetylcholinesterase and butyrylcholinesterase genes.
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Acetylcholinesterase↗
Amplification of butyrylcholinesterase and acetylcholinesterase genes in normal and tumor tissues: putative relationship to organophosphorous poisoning.
Cholinesterases are ubiquitous carboxylesterase type B enzymes capable of hydrolyzing the neurotransmitter acetylcholine which are transiently expressed in multiple germline, embryonic, and tumor cells. The acute poisoning effects of various organophosphorous compounds are generally attributed to their irreversible covalent interaction with cholinesterases and block of their catalytic activities. We have recently found a de novo inheritable amplification of a CHE gene encoding defective butyrylcholinesterase (acylcholine acyl hydrolase; EC 3.1.1.8) in a family under prolonged exposure to the agricultural organophosphorous insecticide methyl parathion. Further analysis revealed that both the CHE and the ACHE genes, encoding acetylcholinesterase (acetylcholine acetyl hydrolase; EC 3.1.1.7), are amplified in leukemias and platelet disorders and that the tumorigenic expression of these genes in ovarian carcinomas is associated with their frequent coamplification in these tumors. The amplification of CHE and ACHE genes in normal and tumor tissues might be analogous to the well-known amplification of other genes encoding target proteins to toxic compounds. As such, it could provide cells a selection advantage when exposed to organophosphorous poisons. Further, since cholinesterases appear to play developmentally important roles in multiple cell types, the amplification and overexpression of their corresponding genes might affect fertility, be related to the progression of various tumor types, and bear upon the ecological and clinical risks involved with the common use of organophosphorous poisons.
Acetylcholinesterase↗
Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G + C-rich attenuating structure.
To study the primary structure of human acetylcholinesterase (AcChoEase; EC 3.1.1.7) and its gene expression and amplification, cDNA libraries from human tissues expressing oocyte-translatable AcChoEase mRNA were constructed and screened with labeled oligodeoxynucleotide probes. Several cDNA clones were isolated that encoded a polypeptide with greater than or equal to 50% identically aligned amino acids to Torpedo AcChoEase and human butyrylcholinesterase (BtChoEase; EC 3.1.1.8). However, these cDNA clones were all truncated within a 300-nucleotide-long G + C-rich region with a predicted pattern of secondary structure having a high Gibbs free energy (-117 kcal/mol) downstream from the expected 5' end of the coding region. Screening of a genomic DNA library revealed the missing 5' domain. When ligated to the cDNA and constructed into a transcription vector, this sequence encoded a synthetic mRNA translated in microinjected oocytes into catalytically active AcChoEase with marked preference for acetylthiocholine over butyrylthiocholine as a substrate, susceptibility to inhibition by the AcChoEase inhibitor BW284C51, and resistance to the BtChoEase inhibitor tetraisopropylpyrophosphoramide. Blot hybridization of genomic DNA from different individuals carrying amplified AcChoEase genes revealed variable intensities and restriction patterns with probes from the regions upstream and downstream from the predicted G + C-rich structure. Thus, the human AcChoEase gene includes a putative G + C-rich attenuator domain and is subject to structural alterations in cases of AcChoEase gene amplification.
Acetylcholinesterase↗
Manipulations of cholinesterase gene expression modulate murine megakaryocytopoiesis in vitro.
Megakaryocytopoiesis was selectively inhibited in cultured murine bone marrow cells by a 15-mer oligodeoxynucleotide complementary to the initiator AUG region in butyrylcholinesterase mRNA. Furthermore, conditioned medium from Xenopus oocytes producing recombinant butyrylcholinesterase stimulated megakaryocytopoiesis. These observations implicate butyrylcholinesterase in megakaryocytopoiesis and suggest application of oligodeoxynucleotides for modulating bone marrow development.
Amino Acid Sequence↗
Acetylcholinesterase and butyrylcholinesterase genes coamplify in primary ovarian carcinomas.
The genes for acetylcholinesterase (ACHE) and butyrylcholinesterase (CHE) are expressed in multiple tumor tissues, including ovarian carcinomas. Both CHE and ACHE genes coamplify in leukemias. To examine the relationship of gene amplification to the expression of these genes in tumors, ACHE and CHE genes and their expression were studied in primary ovarian carcinomas. DNA blot hybridization demonstrated a significant amplification and mutagenesis of both genes in 6 of 11 malignant tumors studied. This was greater or of the same order of magnitude as the amplification of the oncogenes c-rafi, v-sis, and c-fes in these tumors. No amplification was found in normal ovarian tissues or benign ovarian cysts. Xenopus oocyte microinjections, blot and in situ hybridizations, and immuno- and cytochemical staining revealed translatable CHEmRNA and its active protein product in discrete tumor foci. The frequent coamplification in ovarian carcinomas of ACHE and CHE genes implicates cholinesterases in neoplastic growth and/or proliferation.
Acetylcholinesterase↗
Efficient functioning of plant promoters and poly(A) sites in Xenopus oocytes.
Mature Xenopus oocytes were challenged with DNA constructs including plant regulatory elements, namely, the Cauliflower mosaic virus (CaMV) 35S promoter as well as the nopaline synthase (NOS) promoter and polyadenylation signal. The bacterial chloramphenicol acetyl transferase (CAT) was used as a reporter gene. When microinjected into these cells, the plant-derived DNA constructs effectively promoted CAT synthesis in a manner dependent on the presence of the plant promoters and probably also on the polyadenylation signals. Structural studies revealed that the supercoiled structures of the above DNA plasmids were much more active in supporting CAT synthesis in microinjected oocytes than their linear forms, with clear correlation between efficient gene expression and DNA topology. In contrast, the linear forms of these plasmids were considerably more active than the supercoiled ones in transfected plant protoplasts. These findings demonstrate, for the first time, the activity of regulatory elements from plant genes in Xenopus oocytes and shed new light on the specific rules applicable for gene expression in plant and animal cells.
Amino Acid Oxidoreductases↗
Expression and tissue-specific assembly of human butyrylcholine esterase in microinjected Xenopus laevis oocytes.
Cholinesterases represent a ubiquitous, polymorphic family of acetylcholine hydrolyzing enzymes. The multileveled tissue-specific heterogeneity which characterizes these enzymes makes the cholinesterases an appropriate model for studying the mechanisms involved in regulating divergent pathways in protein biogenesis. For this purpose, a cDNA coding for human butyrylcholine esterase (BuChE) was subcloned into the SP 6 transcription vector. Synthetic mRNA transcribed from this construct was microninjected into Xenopus laevis oocytes alone, and in conjunction with poly(A)+ RNAs extracted from human brain or muscle. Injected BuChE-mRNA induced the biosynthesis of a protein exhibiting the catalytic activity, substrate specificity, and sensitivity to selective inhibitors characteristic of native human serum BuChE, and clearly distinct from the related enzyme acetylcholinesterase (AChE). The nascent BuChE was reproducibly distributed into low salt-soluble and detergent-extractable pools. Sucrose gradient analysis demonstrated that the nascent human enzyme was capable of limited subunit assembly, appearing as functional dimeric molecules in both of these fractions. Co-injection with brain or muscle-derived mRNAs facilitated higher order oligomeric assembly. Co-injected brain mRNA induced the appearance of tetramers while co-injected muscle mRNA induced the appearance of an array of heavy molecular forms, including a heavy 16 S form. These results indicate that the molecular determinants which distinguish BuChE from AChE are inherent to its primary amino acid sequence and that additional, tissue-specific protein(s) are involved in the modulation of subunit assembly within particular biological milieues.
Animals↗
[In-situ hybridization on chorionic villi chromosomes].
Novel techniques for the prenatal diagnosis of inherited defects are currently being developed. The long-range aim is to be able to predict precisely, at an early stage of fetal development, the tendency of the fetus to develop multiple genetic, congenital or acquired diseases. We adapted the technique of gene mapping by in-situ hybridization for use with chromosomes from fetal chorionic villi sampling (CVS). Refined mapping of the genes coding for cholinesterase (CHE) in comparison with the haptoglobin and the transferrin receptor genes on CVS chromosomes Nos. 3 and 16 revealed 3 CHE genes in positions 3q21, 3q26, and 16q12. In view of genetic linkage data, at least 2 of these appear to be potentially active. These findings demonstrate that genes, for which molecularly cloned DNA probes are available, may be localized on CVS chromosomes by comparing their localization with that of known genes after in-situ hybridization. The implications for prenatal diagnosis are promising.
Cholinesterases↗
Tissue-specific processing and polarized compartmentalization of clone-produced cholinesterase in microinjected Xenopus oocytes.
1. To approach the involvement of tissue-specific elements in the compartmentalization of ubiquitous polymorphic proteins, immunohistochemical methods were used to analyze the localization of butyrylcholinesterase (BuChE) in Xenopus oocytes microinjected with synthetic BuChEmRNA alone and in combination with tissue-extracted mRNAs. 2. When injected alone BuChEmRNA efficiently directed the synthesis of small membrane-associated accumulations localized principally on the external surface of the oocyte's animal pole. Tunicamycin blocked the appearance of such accumulations, suggesting that glycosylation is involved in the transport of nascent BuChE molecules to the oocyte's surface. Coinjection with brain or muscle mRNA, but not liver mRNA, facilitated the formation of pronounced, tissue-characteristic BuChE aggregates. 3. These findings implicate tissue-specific mRNAs in the assembly of the clone-produced protein and in its nonuniform distribution in the oocyte membrane or extracellular material.
Animals↗
Cholinoceptive properties of human primordial, preantral, and antral oocytes: in situ hybridization and biochemical evidence for expression of cholinesterase genes.
In addition to their well-known involvement in neuromuscular junctions and in brain cholinergic synapses, cholinergic mechanisms have been implicated in the growth and maturation of oocytes in various species. Functional acetylcholine receptors were electrophysiologically demonstrated in amphibian and mammalian oocyte membranes, and activity of the acetylcholine-hydrolyzing enzyme, acetylcholinesterase (AChE), was biochemically measured in the exceptionally big oocytes of the frog Xenopus laevis. However, biochemical methods could not reveal whether AChE was produced within the oocytes themselves or in the surrounding follicle cells. Furthermore, this issue is particularly important for understanding growth and fertilization processes in the much smaller human oocytes, in which the sensitivity of AChE biochemical measurements is far too low to be employed. To resolve this question, a molecular biology approach was combined with biochemical measurements on ovarian extracts and sections. To directly determine whether the human cholinesterase (ChE) genes are transcriptionally active in oocytes, and, if so, at what stages in their development, the presence of ChE mRNA was pursued. For this purpose frozen ovarian sections were subjected to in situ hybridization using 35S-labeled human ChE cDNA. Highly pronounced hybridization signals were localized within oocytes in primordial, preantral, and antral follicles, but not in other ovarian cell types, demonstrating that within the human ovary ChE mRNA is selectively synthesized in viable oocytes at different developmental stages.(ABSTRACT TRUNCATED AT 250 WORDS)
Adult↗
Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: correlation with various leukemias and abnormal megakaryocytopoiesis.
To study the yet unknown role of the ubiquitous family of cholinesterases (ChoEases) in developing blood cells, the recently isolated cDNAs encoding human acetylcholinesterase (AcChoEase; acetylcholine acetylhydrolase, EC 3.1.1.7) and butyrylcholinesterase (BtChoEase; cholinesterase; acylcholine acylhydrolase, EC 3.1.1.8) were used in blot hybridization with peripheral blood DNA from various leukemic patients. Hybridization signals (10- to 200-fold intensified) and modified restriction patterns were observed with both cDNA probes in 4 of the 16 leukemia DNA preparations examined. These reflected the amplification of the corresponding AcChoEase and BtChoEase genes (ACHE and CHE) and alteration in their structure. Parallel analysis of 30 control samples revealed nonpolymorphic, much weaker hybridization signals for each of the probes. In view of previous reports on the effect of acetylcholine analogs and ChoEase inhibitors in the induction of megakaryocytopoiesis and production of platelets in the mouse, we further searched for such phenomena in nonleukemic patients with platelet production disorders. Amplifications of both ACHE and CHE genes were found in 2 of the 4 patients so far examined. Pronounced coamplification of these two related but distinct genes in correlation with pathological production of blood cells suggests a functional role for members of the ChoEase family in megakaryocytopoiesis and raises the question whether the coamplification of these genes could be causally involved in the etiology of hemocytopoietic disorders.
Acetylcholinesterase↗
De novo amplification within a "silent" human cholinesterase gene in a family subjected to prolonged exposure to organophosphorous insecticides.
A 100-fold DNA amplification in the CHE gene, coding for serum butyrylcholinesterase (BtChoEase), was found in a farmer expressing the "silent" CHE phenotype. Individuals homozygous for this gene display a defective serum BtChoEase and are particularly vulnerable to poisoning by agricultural organophosphorous insecticides, to which all members of this family had long been exposed. DNA blot hybridization with regional BtChoEase cDNA probes suggested that the amplification was most intense in regions encoding central sequences within BtChoEase cDNA, whereas distal sequences were amplified to a much lower extent. This is in agreement with the "onion skin" model, based on amplification of genes in cultured cells and primary tumors. The amplification was absent in the grandparents but present at the same extent in one of their sons and in a grandson, with similar DNA blot hybridization patterns. In situ hybridization experiments localized the amplified sequences to the long arm of chromosome 3, close to the site where we previously mapped the CHE gene. Altogether, these observations suggest that the initial amplification event occurred early in embryogenesis, spermatogenesis, or oogenesis, where the CHE gene is intensely active and where cholinergic functioning was indicated to be physiologically necessary. Our findings demonstrate a de novo amplification in apparently healthy individuals within an autosomal gene producing a target protein to an inhibitor. Its occurrence in two generations from a family under prolonged exposure to parathion indicates that organophosphorous poisons may be implicated in previously unforeseen long-term ecological effects.
Butyrylcholinesterase↗
Gene mapping on chorionic villi chromosomes by hybridization in situ: localization of cholinesterase cDNA binding sites to chromosomes 3q21, 3q26-ter and 16q21.
To adapt the in-situ hybridization approach for use in very early fetal development, comparative in-situ hybridization was initiated on chromosomes from chorionic villus sampling (CVS). An additional aim was to refine the localization of the chromosomal sites binding butyrylcholinesterase (BuChE) cDNA by mapping them in parallel to previously mapped genes of close locations. BuChEcDNA was previously found to bind to the long arms of lymphocyte chromosomes 3 and 16, with a wide distribution of grains suggesting two separate sites on chromosome 3. When labelled with 35S and hybridized with CVS chromosomes, BuChEcDNA was bound to three distinct sites, designated CHEL1, CHEL2 and CHEL3. These peaked at 3q21, 3q26-ter and 16q21 respectively. Parallel hybridization with the cDNA encoding transferrin receptor (TFRC) refined its localization to 3q29, in agreement with previously published results and in a distal position to CHEL2, whereas haptoglobin cDNA (HPcDNA) was correctly mapped at 16q24, distal to CHEL3. In view of the published genetic linkage between the CHEL1 locus and the transferrin TF gene on 3q25, this study suggests that one of the three sites carrying BuChE-coding sequences, namely CHEL2, harbours the functional CHEL1 gene. Thus, in-situ hybridization provides a rapid and precise method for the localization of genes on CVS chromosomes, in comparison with known DNA markers.
Binding Sites↗
trans activation of rat phosphoenolpyruvate carboxykinase (GTP) gene expression by micro-coinjection of rat liver mRNA in Xenopus laevis oocytes.
To study the liver-specific trans activation of the rat phosphoenolpyruvate carboxykinase (PEPCK) gene, the PEPCK promoter was linked to a reporter gene and was microinjected into Xenopus laevis oocytes alone or in conjunction with rat liver poly(A)+ RNA. The rat liver mRNA markedly enhanced the expression of the PEPCK-chimeric construct. This effect appeared to be sequence specific, as it was dependent on the presence of the intact promoter. Moreover, the RNA effect was limited to mRNA preparations from PEPCK-expressing tissues only. Finally, microinjection of size-fractionated liver mRNA revealed that the trans-acting factor(s) is encoded by RNA of 1,600 to 2,000 nucleotides, providing a direct bioassay for the gene(s) involved in this tissue-specific trans-activation process.
Animals↗
Definition, at the molecular level, of a thyroglobulin-acetylcholinesterase shared epitope: study of its pathophysiological significance in patients with Graves' ophthalmopathy.
The nature of the putative autoantigen in Graves' ophthalmopathy (Go) remains an enigma but the sequence similarity between thyroglobulin (Tg) and acetylcholinesterase (ACHE) provides a rationale for epitopes which are common to the thyroid gland and the eye orbit. In an attempt to define the shared epitope, we have screened a lambda gt 11 human thyroid cDNA library using a polyclonal antibody to Torpedo ACHE and isolated two clones, which upon sequencing, were shown to contain Tg segments, corresponding to portions of the C terminal part of the molecule which has a high similarity with ACHE. Having demonstrated the existence of an epitope common to Tg and ACHE, the clones have been further tested and found to be positive in lysis plaque assays with 1/10 sera from patients with Hashimoto's thyroiditis (HT), 8/8 from patients with Graves' ophthalmopathy and 0/8 normal sera. We have investigated the physiological significance of this common epitope by in situ immunolocalization studies in which the polyclonal antibody to Torpedo ACHE (which was used for screening the library) and immunoglobulins (Igs) from 6 Go patients tested were shown to bind to end plate regions of human foetal muscle fibres which were concurrently shown to be rich in cholinesterase activity: Igs from 3 normal individuals and 2 patients with Hashimoto's thyroiditis did not bind. The results demonstrate and characterize an epitope which is common to Tg and ACHE and show that Go patients Igs contain antibodies which bind to muscle end plates rich in cholinesterase. The significance of these findings to the pathogenesis of Go is discussed.
Acetylcholinesterase↗
Sequence similarities between human acetylcholinesterase and related proteins: putative implications for therapy of anticholinesterase intoxication.
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Acetylcholinesterase↗
Modified properties of serum cholinesterases in primary carcinomas.
Cholinesterases were characterized in the serum of 77 treated and 11 untreated patients having primary carcinomas of various tissue origins and 21 healthy volunteers which served as controls. In most of the samples, pseudocholinesterase (BuChE) accounted for almost all cholinesterase (ChE) activity and was inhibited by the organophosphorous poison tetraisopropyl pyrophosphoramide (iso-OMPA). In samples from the tumor-bearing patients, ChE degraded 733 +/- 59 nmole acetylcholine/h/mg protein, lower than the 960 +/- 175 nmole/hour/mg levels measured in controls. Tumor serum ChE exhibited elevated sensitivity to 1,5-bis-(4-allyldimethyl ammonium phenyl)-pentan-3-one dibromide (BW), the selective bisquaternary inhibitor of "true" acetylcholinesterase (AChE), with no correlation to age, sex, staging of tumor, presence of metastases or the specific treatment protocol, and with a different distribution pattern from the decrease in ChE specific activity or the sensitivity to iso-OMPA. In sucrose gradients, ChE sedimented as 12S in controls whereas in tumor serum samples from treated patients an additional component of 6 to 7 S, inhibited by both iso-OMPA and BW, also was detected. However, the ChE activity in serum of patients with diagnosed carcinomas before surgery and medical treatment appeared to be nondistinguishable from controls. These findings suggest that the modified properties of serum cholinesterases in carcinoma patients are not the result of the tumor itself, but that the common therapy protocols used in the treatment of primary carcinomas may cause the appearance of soluble ChE activity with properties of both AChE and BuChE, which accumulates in the serum.
Adult↗