Identification of functional murine adenosine deaminase cDNA clones by complementation in Escherichia coli.
Total poly(A+) RNA derived from a mouse cell line with amplified adenosine deaminase genes was used as template to synthesize double-stranded cDNA. The cDNAs were inserted into the PstI site of the beta-lactamase gene in plasmid pBR322 following G-C tailing. After transformation into adenosine deaminase-deficient Escherichia coli hosts, recombinant plasmids containing functional murine adenosine deaminase cDNAs were identified by selecting for functional complementation. Analysis of plasmids containing functional adenosine deaminase cDNA sequences strongly suggested that adenosine deaminase expression resulted mainly from beta-lactamase/adenosine deaminase fusion proteins even when the adenosine deaminase codons were out-of-frame with respect to the beta-lactamase gene codons upstream. The nucleotide sequence of a 1.65-kilobase pair cDNA insert in one of the functional recombinant clones was determined and found to contain a 1056-nucleotide open reading frame. When this 1056-nucleotide open reading frame was inserted into a mammalian expression vector and introduced into monkey kidney cells, a high level of authentic mouse adenosine deaminase was produced. Nucleic acid blot analysis using a full-length adenosine deaminase cDNA clone as probe revealed that the mouse adenosine deaminase structural gene was at least 21 kilobase pairs in size and encoded three polyadenylated mRNAs. Analysis of the cDNA library from which the functional clones were isolated suggested that this approach of cloning functional mammalian adenosine deaminase cDNA clones by genetic complementation of enzyme-deficient bacteria could be accomplished even if the abundance of the adenosine deaminase mRNA sequences were as low as approximately 0.001%.
Yeung
C Y
CY
Ingolia
D E
DE
Roth
D B
DB
Shoemaker
C
C
Al-Ubaidi
M R
MR
Yen
J Y
JY
Ching
C
C
Bobonis
C
C
Kaufman
R J
RJ
Kellems
R E
RE
D000243:Adenosine Deaminase / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics*; D005784:Gene Amplification; D005796:Genes; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007668:Kidney; D051379:Mice; D009693:Nucleic Acid Hybridization; D009700:Nucleoside Deaminases / Q000235:genetics*; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D013937:Thymidine Kinase / Q000235:genetics
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D013937:Thymidine Kinase; D009700:Nucleoside Deaminases; D000243:Adenosine Deaminase