1. Various hybridization approaches were employed to investigate structural and chromosomal interrelationships between the human cholinesterase genes CHE and ACHE encoding the polymorphic, closely related, and coordinately regulated enzymes having butyrylcholinesterase (BCHE) and acetylcholinesterase (AChE) activities. 2. Homologous cosmid recombination with a 190-base pair 5' fragment from BCHEcDNA resulted in the isolation of four overlapping cosmid clones, apparently derived from a single gene with several introns. The Cosmid CHEDNA included a 700-base pair fragment known to be expressed at the 3' end of BCHEcDNA from nervous system tumors and which has been mapped by in situ hybridization to the unique 3q26-ter position. In contrast, cosmid CHEDNA did not hybridize with full-length AChEcDNA, proving that the complete CHE gene does not include AChE-encoding sequences either in exons or in its introns. 3. The chromosomal origin of BCHE-encoding sequences was further examined by two unrelated gene mapping approaches. Filter hybridization with DNA from human/hamster hybrid cell lines revealed BCHEcDNA-hybridizing sequences only in cell lines including human chromosome 3. However, three BCHEcDNA-homologous sequences were observed at chromosomal positions 3q21, 3q26-ter, and 16q21 by a highly stringent in situ hybridization protocol, including washes at high temperature and low salt. 4. These findings stress the selectivity of cosmid recombination and chromosome blots, raise the possibility of individual differences in BCHEcDNA-hybridizing sequences, and present an example for a family highly similar proteins encoded by distinct, nonhomologous genes.
Gene expression in chorionic villi may be particularly vulnerable to environmental exposure to poisonous substances. To reveal villus gene products which are thus subject to poisoning, molecular cloning was employed. A single sample of apparently normal chorionic villi (approximately 40 mg, from 9 weeks' gestation) was microscopically dissected to ensure purity of fetal tissue. Total RNA was extracted by isothiocyanate and directly employed for reverse transcription. A chorionic villus cDNA library was constructed from this preparation in the phage vector lambda gt10 and contained 60,000 independent recombinants. In the present study, this cDNA library was screened with labelled cDNA probes encoding human butyrylcholinesterase (BCHE) and acetylcholinesterase (ACHE). Nine BCHEcDNA clones were isolated out of 1.6 x 10(6) phages (5.7 x 10(-6) of screened recombinants) and exhibited similar restriction patterns to those observed for BCHEcDNA from other human tissues. In contrast, no ACHEcDNA clones could be found in 4.0 x 10(6) screened phages (less than 2.5 x 10(-6) of recombinants). These findings demonstrate efficient transcription (similar to fetal brain levels) from the BCHE gene but not from the ACHE gene in chorionic villi, and support the notion that BCHE is involved in chorionic villus growth and development.
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.
