Title: Multiple nuclear proteins bind a novel cis-acting element that regulates the muscle-specific expression of the mouse nicotinic acetylcholine receptor alpha-subunit gene Dennis P, Prody CA Ref: DNA & Cell Biology, 16:1099, 1997 : PubMed
Expression of the nicotinic acetylcholine receptor (AChR) is transcriptionally regulated during the development of vertebrate striated muscle. To better define regulatory elements involved in this process, site-directed mutations were made in the gene's 86 bp muscle specific enhancer. Transient expression assays in skeletal muscle C2C12 cells indicated that all three E-boxes, plus a novel sequence outside the E-boxes, are necessary for full activity of the AChR gene in myotubes. Gel mobility shift assays demonstrated that mutations in the non-E-box sequence disrupted the formation of two DNA/protein complexes while not affecting myoD binding. Methylation interference footprinting confirmed that the complexes form at nucleotides within the mutated region, and also include part of the central E-box. UV crosslinking of nuclear proteins to a DNA probe identified five proteins of 125, 81, 55, 42, and 35 kDa that bind to this region; with the 125 kDa protein being differentially bound in U.V. crosslink assays during the transition from myoblasts to myotubes. These data suggest that interactions between this DNA element and the five proteins contribute to the transcriptional control of the AChR alpha-subunit gene expression during the differentiation of skeletal muscle.
Mechanisms triggering the commitment of pluripotent bone marrow stem cells to differentiated lineages such as mononuclear macrophages or multinucleated megakaryocytes are still unknown, although several lines of evidence suggested correlation between cholinergic signaling and hematopoietic differentiation. We now present cloning of a cDNA coding for CHED (cholinesterase-related cell division controller), a human homolog of the Schizosaccharomyces pombe cell division cycle 2 (cdc2)-like kinases, universal controllers of the mitotic cell cycle. Library screening, RNA blot hybridization, and direct PCR amplification of cDNA reverse-transcribed from cellular mRNA revealed that CHED mRNA is expressed in multiple tissues, including bone marrow. The CHED protein includes the consensus ATP binding and phosphorylation domains characteristic of kinases, displays 34-42% identically aligned amino acid residues with other cdc2-related kinases, and is considerably longer at its amino and carboxyl termini. An antisense oligodeoxynucleotide designed to interrupt CHED's expression (AS-CHED) significantly reduced the ratio between CHED mRNA and actin mRNA within 1 hr of its addition to cultures, a reduction that persisted for 4 days. AS-CHED treatment selectively inhibited megakaryocyte development in murine bone marrow cultures but did not prevent other hematopoietic pathways, as evidenced by increasing numbers of mononuclear cells. An oligodeoxynucleotide blocking production of the acetylcholine-hydrolyzing enzyme, butyrylcholinesterase, displayed a similar inhibition of megakaryocytopoiesis. In contrast, an oligodeoxynucleotide blocking production of the human 2Hs cdc2 homolog interfered with production of the human 2Hs cdc2 homolog interfered with cellular proliferation without altering the cell-type composition of these cultures. Therefore, these findings strengthen the link between cholinergic signaling and cell division control in hematopoiesis and implicate both CHED and cholinesterases in this differentiation process.
Title: The 5'-flanking region of the mouse muscle nicotinic acetylcholine receptor beta subunit gene promotes expression in cultured muscle cells and is activated by MRF4, myogenin and myoD Prody CA, Merlie JP Ref: Nucleic Acids Research, 20:2367, 1992 : PubMed
The expression of the nicotinic acetylcholine receptor (AChR) in vertebrate striated muscle is regulated both during development by nerve-evoked muscle activity and by local factors released or associated with the nerve ending. The expression pattern of AChR is achieved by coordinate regulation of four embryonic subunit mRNAs, alpha, beta, gamma and delta. We have taken the approach of identifying the similarities and differences among cis-acting regulatory elements of AChR genes to gain a better understanding of these mechanisms. Thus, to begin to define DNA sequences necessary for the transcriptional regulation of the mouse beta AChR gene, we have analyzed its 5'-flanking region. Primer extension and RNAase protection analyses showed that transcription initiates at one major and two minor sites, all of which are close to the translational initiation site. Using plasmids in which segments of the 5'-flanking region were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, we have demonstrated that 150 bp of the 5'-flanking region is active in C2 myotubes but not C2 myoblasts or NIH3T3 fibroblasts. This region contains a putative binding site for myoD, and when linked to CAT was transactivated by the muscle regulatory factors myoD, myogenin, and MRF4. Thus, a 150 bp sequence of the beta-subunit gene contains information necessary for developmental specificity and responsiveness to myogenic factors.
Title: A developmental and tissue-specific enhancer in the mouse skeletal muscle acetylcholine receptor alpha-subunit gene regulated by myogenic factors Prody CA, Merlie JP Ref: Journal of Biological Chemistry, 266:22588, 1991 : PubMed
The expression of the nicotinic acetylcholine receptor (AChR) in vertebrate striated muscle is regulated both during development and in response to nerve-evoked muscle activity. To define DNA sequences necessary for the transcriptional regulation of the mouse alpha-subunit AChR gene, we have isolated and analyzed the alpha-gene 5'-flanking region. Primer extension and RNase protection analysis showed that transcription initiates at 2 major and 12 minor sites close to the translational initiation site. Using a series of plasmids in which segments of the 5'-flanking region were linked to the bacterial chloramphenicol acetyltransferase (CAT) gene, we have defined an 86-base pair enhancer sequence that is active in C2 myotubes but not in C2 myoblasts or NIH3T3 fibroblasts. This enhancer contains three putative binding sites for myoD1, and the 5'-upstream regions linked to CAT were transactivated by the muscle regulatory factors, myoD1, and myogenin. Transactivation by MRF4 differed with the specific alpha-subunit construct tested. Whereas the alpha-subunit CAT constructs containing both the homologous as well as the heterologous myosin light chain 1 promoter were transactivated by myoD1 and myogenin, only the constructs containing their homologous promoter were transactivated by MRF4. Thus, an 86-base pair sequence of the alpha-subunit gene contains the information necessary for developmental specificity and responsiveness to myogenic factors.
Title: Expression of alternatively terminated unusual human butyrylcholinesterase messenger RNA transcripts, mapping to chromosome 3q26-ter, in nervous system tumors Gnatt A, Prody CA, Zamir R, Lieman-Hurwitz J, Zakut H, Soreq H Ref: Cancer Research, 50:1983, 1990 : PubMed
To study the molecular origin of the altered regulation of butyrylcholinesterase (BCHE) in nervous system tumors, BCHE complementary DNA (cDNA) sequences from human glioblastoma and neuroblastoma cDNA libraries were compared with BCHE cDNAs from normal fetal and adult tissues. A single 2.6-kilobase BCHE cDNA sequence was found in all normal tissues, whereas an additional alternatively terminated BCHE cDNA clone was found in both tumor libraries. The tumor-specific cDNA contained a 3',0.7-kilobase nontranslatable extension, as well as several nucleotide alterations in the normal polyadenylation site. Single-base mutations in the coding region of this unusual BCHE cDNA infer two amino acid substitutions: Asp70----Gly and Ser425----Pro. The Asp70----Gly change has recently been implicated with "atypical" BCHE, which is deficient in its capacity to hydrolyze succinylcholine. The 3.6-kilobase mRNA was less abundant in RNA blot hybridization than the 2.6-kilobase mRNA, which is in agreement with the low ratios between the 3.6- and 2.6-kilobase BCHE cDNA clones in glioblastoma and neuroblastoma libraries. Furthermore, size fractionation and microinjection of glioblastoma polyadenylated RNA, followed by enzyme activity and selective inhibition measurements, demonstrated two peaks of functional BCHE mRNA, the heavier one probably reflecting the longer transcripts. Chromosomal mapping of the 0.7-kilobase 3' fragment by in situ hybridization localized it to a unique 3q26-ter position, where we recently found an inheritably amplified "silent" defective CHE gene in a family exposed to the cholinesterase inhibitor methyl parathion. Our findings confirm previous genetic linkage mapping of the functional CHE gene to the 3q26-ter position and demonstrate that extended functional mRNA transcripts encoding a BCHE form with two modified amino acids are produced from this gene in glioblastoma and neuroblastoma cells.
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.
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.
Title: Coamplification of human acetylcholinesterase and butyrylcholinesterase genes in blood cells: correlation with various leukemias and abnormal megakaryocytopoiesis Lapidot-Lifson Y, Prody CA, Ginzberg D, Meytes D, Zakut H, Soreq H Ref: Proceedings of the National Academy of Sciences of the United States of America, 86:4715, 1989 : PubMed
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.
Title: De novo amplification within a silent human cholinesterase gene in a family subjected to prolonged exposure to organophosphorous insecticides Prody CA, Dreyfus PA, Zamir R, Zakut H, Soreq H Ref: Proceedings of the National Academy of Sciences of the United States of America, 86:690, 1989 : PubMed
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.
Title: Sequence similarities between human acetylcholinesterase and related proteins: putative implications for therapy of anticholinesterase intoxication Soreq H, Prody CA Ref: Progress in Clinical & Biological Research, 289:347, 1989 : PubMed
Title: Cross-homologies and structural differences between human cholinesterases revealed by antibodies against cDNA-produced human butyrylcholinesterase peptides Dreyfus PA, Zevin-Sonkin D, Seidman S, Prody CA, Zisling R, Zakut H, Soreq H Ref: Journal of Neurochemistry, 51:1858, 1988 : PubMed
To study the polymorphism of human cholinesterases (ChEs) at the levels of primary sequence and three-dimensional structure, a fragment of human butyrylcholinesterase (BCHE) cDNA was subcloned into the pEX bacterial expression vector and its polypeptide product analyzed. Immunoblot analysis revealed that the clone-produced BCHE peptides interact specifically with antibodies against human and Torpedo acetylcholinesterase (AChE). Rabbit polyclonal antibodies prepared against the purified clone-produced BCHE polypeptides interacted in immunoblots with denatured serum BCHE as well as with purified and denatured erythrocyte AChE. In contrast, native BCHE tetramers from human serum, but not AChE dimers from erythrocytes, interacted with these antibodies in solution to produce antibody-enzyme complexes that could be precipitated by second antibodies and that sedimented faster than the native enzyme in sucrose gradient centrifugation. Furthermore, both AChE and BCHE dimers from muscle extracts, but not BCHE tetramers from muscle, interacted with these antibodies. To reveal further whether the anti-cloned BCHE antibodies would interact in situ with ChEs in the neuromuscular junction, bundles of muscle fibers were microscopically dissected from the region in fetal human diaphragm that is innervated by the phrenic nerve. Muscle fibers incubated with the antibodies and with 125I-Protein A were subjected to emulsion autoradiography, followed by cytochemical ChE staining. The anti-cloned BCHE antibodies, as well as anti-Torpedo AChE antibodies, created patches of silver grains in the muscle endplate region stained for ChE, under conditions where control sera did not. These findings demonstrate that the various forms of human AChE and BCHE in blood and in neuromuscular junctions share sequence homologies, but also display structural differences between distinct molecular forms within particular tissues, as well as between similarly sedimenting molecular forms from different tissues.
Title: Isolation and characterization of full-length cDNA clones coding for cholinesterase from fetal human tissues Prody CA, Zevin-Sonkin D, Gnatt A, Goldberg O, Soreq H Ref: Proceedings of the National Academy of Sciences of the United States of America, 84:3555, 1987 : PubMed
To study the primary structure and regulation of human cholinesterases, oligodeoxynucleotide probes were prepared according to a consensus peptide sequence present in the active site of both human serum pseudocholinesterase (BtChoEase; EC 3.1.1.8) and Torpedo electric organ "true" acetylcholinesterase (AcChoEase; EC 3.1.1.7). Using these probes, we isolated several cDNA clones from lambda gt10 libraries of fetal brain and liver origins. These include 2.4-kilobase cDNA clones that code for a polypeptide containing a putative signal peptide and the N-terminal, active site, and C-terminal peptides of human BtChoEase, suggesting that they code either for BtChoEase itself or for a very similar but distinct fetal form of cholinesterase. In RNA blots of poly(A)+ RNA from the cholinesterase-producing fetal brain and liver, these cDNAs hybridized with a single 2.5-kilobase band. Blot hybridization to human genomic DNA revealed that these fetal BtChoEase cDNA clones hybridize with DNA fragments of the total length of 17.5 kilobases, and signal intensities indicated that these sequences are not present in many copies. Both the cDNA-encoded protein and its nucleotide sequence display striking homology to parallel sequences published for Torpedo AcChoEase. These findings demonstrate extensive homologies between the fetal BtChoEase encoded by these clones and other cholinesterases of various forms and species.
Cholinesterases are serine esterases that rapidly hydrolyze the neurotransmitter acetylcholine. In humans, cholinesterases exhibit extensive polymorphism in terms of their substrate specificity, sensitivity to selective inhibitors, hydrophobicity, and cellular as well as subcellular localization. It is not yet known whether the various cholinesterase forms originate from different genes or are products of posttranscriptional and posttranslational processing. The extent to which these enzyme forms are homologous in their amino acid sequence is also not known. However, a consensus organophosphate-binding hexapeptide sequence Phe-Gly-Glu-Ser-Ala-Gly was found both in "true" acetylcholinesterase from the electric organ of Torpedo [McPhee-Quigley et al: J Biol Chem 260:12185-12189, 1985] and in "pseudocholinesterase" (butyrylcholinesterase) from human serum [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter pp 5-12, 1984], suggesting that this region in the protein is conserved in all cholinesterases. Based on this common sequence, we prepared synthetic oligodeoxynucleotides and used them as labeled probes to screen a cDNA library from fetal human brain mRNA, cloned in lambda gt10 phages. A cDNA clone of 770 nucleotides in length was isolated. It contains an open reading frame terminating with the sequence Ser-Val-Thr-Leu-Phe-Gly-Glu-Ser-Ala-Gly-Ala-Ala, which includes the consensus hexapeptide used for designing the DNA probe. Furthermore, the sequence of this 12-amino acid peptide is identical to the sequence reported for the organophosphate binding site of human serum pseudocholinesterase [Lockridge: "Cholinesterases--Fundamental and Applied Aspects." New York: de Gruyter, pp 5-12, 1984]. These findings confirm that the isolated clone is indeed part of a human cholinesterase cDNA.
