Cloning, sequencing, and expression of cDNA for human. beta. -glucuronidase
The authors report here the cDNA sequence for human placental ..beta..-glucuronidase (..beta..-D-glucuronoside glucuronosohydrolase, EC 3.2.1.31) and demonstrate expression of the human enzyme in transfected COS cells. They also sequenced a partial cDNA clone from human fibroblasts that contained a 153-base-pair deletion within the coding sequence and found a second type of cDNA clone from placenta that contained the same deletion. Nuclease S1 mapping studies demonstrated two types of mRNAs in human placenta that corresponded to the two types of cDNA clones isolated. The NH/sub 2/-terminal amino acid sequence determined for human spleen ..beta..-glucuronidase agreed with that inferred from the DNA sequence of the two placental clones, beginning at amino acid 23, suggesting a cleaved signal sequence of 22 amino acids. When transfected into COS cells, plasmids containing either placental clone expressed an immunoprecipitable protein that contained N-linked oligosaccharides as evidenced by sensitivity to endoglycosidase F. However, only transfection with the clone containing the 153-base-pair segment led to expression of human ..beta..-glucuronidase activity. These studies provide the sequence for the full-length cDNA for human ..beta..-glucuronidase, demonstrate the existence of two populations of mRNA for ..beta..-glucuronidase in human placenta, only one of which specifies a catalytically active enzyme, and illustrate the importance of expression studies in verifying that a cDNA is functionally full-length.
- Research Organization:
- St. Louis Univ. School of Medicine, MO
- OSTI ID:
- 6022997
- Journal Information:
- Proc. Natl. Acad. Sci. U.S.A.; (United States), Vol. 84:3
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
62 RADIOLOGY AND NUCLEAR MEDICINE
GLUCURONIDASE
AMINO ACID SEQUENCE
GENE REGULATION
RECOMBINANT DNA
DNA SEQUENCING
DNA-CLONING
RNA
AUTORADIOGRAPHY
FIBROBLASTS
HYBRIDIZATION
MAN
METHIONINE
PHOSPHORUS 32
PLACENTA
SPLEEN
SULFUR 35
AMINO ACIDS
ANIMAL CELLS
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CARBOXYLIC ACIDS
CLONING
CONNECTIVE TISSUE CELLS
DAYS LIVING RADIOISOTOPES
DNA
DRUGS
ENZYMES
EVEN-ODD NUCLEI
FETAL MEMBRANES
GLYCOSYL HYDROLASES
HYDROLASES
ISOTOPES
LIGHT NUCLEI
LIPOTROPIC FACTORS
MAMMALS
MEMBRANES
MOLECULAR STRUCTURE
NUCLEI
NUCLEIC ACIDS
O-GLYCOSYL HYDROLASES
ODD-ODD NUCLEI
ORGANIC ACIDS
ORGANIC COMPOUNDS
ORGANIC SULFUR COMPOUNDS
ORGANS
PHOSPHORUS ISOTOPES
PRIMATES
RADIOISOTOPES
SOMATIC CELLS
STRUCTURAL CHEMICAL ANALYSIS
SULFUR ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques
550601 - Medicine- Unsealed Radionuclides in Diagnostics