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Title: A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene

Abstract

OTC is a mitochondrial enzyme catalyzing the formation of citrulline from carbamyl phosphate and ornithine. X-linked deficiency of OTC is the most prevalent genetic defect of ureagenesis. Mutations and polymorphisms in the OTC gene identified in deficient patients have indicated the occurrence of many family-specific, unique alleles. Due to the low frequency of recurrent mutations, distinguishing between deleterious mutations and polymorphisms is difficult. Using a human OTC gene containing plasmid driven by a tac promoter, we have devised a simple and efficient method for expressing mutations in the mature human OTC enzyme. To demonstrate this method, PCR engineered site-directed mutagenesis was employed to generated cDNA fragments which contained either the R151Q or R277W known mutations found in patients with neonatal and late-onset OTC deficiency, respectively. The normal allele for each mutation was also generated by an identical PCR procedure. Digestion with Bgl II- and Sty I-generated mutant and normal replacement cassettes containing the respective mutant and wild type sequences. Upon transformation of JM109 E.coli cells, OTC enzymatic activity was measured at log and stationary phases of growth using a radiochromatographic method. The R141Q mutation abolished enzymatic activity (<0.02% of normal), whereas the R277W mutation expressed partial activity (2.3% of normal).more » In addition, a PCR-generated mutation, A280V, resulted in 73% loss of catalytic activity. This OTC expression system is clinically applicable for distinguishing between mutations and polymorphisms, and it can be used to investigate the effects of mutations on various domains of the OTC gene.« less

Authors:
; ;  [1]
  1. Univ. of Minnesota, Mineapolis (United States)
Publication Date:
OSTI Identifier:
134757
Report Number(s):
CONF-941009-
Journal ID: AJHGAG; ISSN 0002-9297; TRN: 95:005313-1496
Resource Type:
Journal Article
Resource Relation:
Journal Name: American Journal of Human Genetics; Journal Volume: 55; Journal Issue: Suppl.3; Conference: 44. annual meeting of the American Society of Human Genetics, Montreal (Canada), 18-22 Oct 1994; Other Information: PBD: Sep 1994
Country of Publication:
United States
Language:
English
Subject:
55 BIOLOGY AND MEDICINE, BASIC STUDIES; GENE MUTATIONS; BIOLOGICAL MODELS; GENES; MUTATION FREQUENCY; ENZYMES; ENZYME ACTIVITY; UROGENITAL SYSTEM DISEASES; AGE DEPENDENCE; PATIENTS; HEREDITARY DISEASES; POLYMERASE CHAIN REACTION; EFFICIENCY; MITOCHONDRIA; ORNITHINE; HUMAN X CHROMOSOME; GENETICS; MUTAGENESIS; ESCHERICHIA COLI

Citation Formats

Tuchman, M., McCann, M.T., and Qureshi, A.A. A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene. United States: N. p., 1994. Web.
Tuchman, M., McCann, M.T., & Qureshi, A.A. A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene. United States.
Tuchman, M., McCann, M.T., and Qureshi, A.A. Thu . "A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene". United States.
@article{osti_134757,
title = {A bacterial model for expression of mutations in the human ornithine transcarbamylase (OTC) gene},
author = {Tuchman, M. and McCann, M.T. and Qureshi, A.A.},
abstractNote = {OTC is a mitochondrial enzyme catalyzing the formation of citrulline from carbamyl phosphate and ornithine. X-linked deficiency of OTC is the most prevalent genetic defect of ureagenesis. Mutations and polymorphisms in the OTC gene identified in deficient patients have indicated the occurrence of many family-specific, unique alleles. Due to the low frequency of recurrent mutations, distinguishing between deleterious mutations and polymorphisms is difficult. Using a human OTC gene containing plasmid driven by a tac promoter, we have devised a simple and efficient method for expressing mutations in the mature human OTC enzyme. To demonstrate this method, PCR engineered site-directed mutagenesis was employed to generated cDNA fragments which contained either the R151Q or R277W known mutations found in patients with neonatal and late-onset OTC deficiency, respectively. The normal allele for each mutation was also generated by an identical PCR procedure. Digestion with Bgl II- and Sty I-generated mutant and normal replacement cassettes containing the respective mutant and wild type sequences. Upon transformation of JM109 E.coli cells, OTC enzymatic activity was measured at log and stationary phases of growth using a radiochromatographic method. The R141Q mutation abolished enzymatic activity (<0.02% of normal), whereas the R277W mutation expressed partial activity (2.3% of normal). In addition, a PCR-generated mutation, A280V, resulted in 73% loss of catalytic activity. This OTC expression system is clinically applicable for distinguishing between mutations and polymorphisms, and it can be used to investigate the effects of mutations on various domains of the OTC gene.},
doi = {},
journal = {American Journal of Human Genetics},
number = Suppl.3,
volume = 55,
place = {United States},
year = {Thu Sep 01 00:00:00 EDT 1994},
month = {Thu Sep 01 00:00:00 EDT 1994}
}