The Characterization of Genomic Human Angiotensinogen Clones by Restriction Mapping
Schadewald, Susan A.
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Human genomic angiotensinogen has been cloned into the EcoRI site of lambda Charon 4A in the laboratory of Dr. K. R. Marotti (The Upjohn Company) in order to study the biosynthesis of angiotensinogen and its possible role in the pathogenesis of human hypertension. The purpose of this study was to characterize the four human genomic angiotensinogen clones by restriction endonuclease cleavaqe analysis and hybridizing experiments. The four clones have been labeled 5, 7, 13, and 14. Clones 5, 7, and 13 were determined to be identical fragments of the angiotensinogen gene. Althouqh clone 14 includes the same fragment as the other clones, it extends approximately 1100 nucleotides more at the 5'- end. The total length of the angiotensinogen gene fragment of clones 5, 7, and 13 is approximately 3700 base pairs, while that of clone 14 is approximately 4800 basepairs long. Only the 3'- end of the human angiotensinogen cDNA, of 1571 base pairs, hybridizes with the four clones, because only the PstI fragments of the cDNA from nucleotides 589 to 1213 (624 base pairs) and from 1213 to 1571 (358 base pairs) were found to hybridize with the clones. It was also determined that the restriction enzymes BamHI, BqlII, EcoRV, HindIII and PvuI have no cleavage sites within any of the four clones. Two different, equally plausible restriction maps for each of clones 13 and 14 were constructed incorporating the cleavage sites of the enzymes EcoRI, PstI, XmnI, BqlI and StuI. These restriction maps will enable the specific fragmentation of the clones as a primary approach to determining the sequence of the nucleotides making up the human angiotensinogen gene.