Isolation, characterization, and expression in Escherichia coli of a cDNA encoding rat heme oxygenase-2.
In a recent study (Cruse, I., and Maines, M.D. (1988) J. Biol. Chem. 263, 3348-3353), we reported the isolation of a small cDNA fragment encoding a portion of heme oxygenase-2 through immunological screening of a rat testis cDNA library in lambda gt11. We have now used this 274-base pair (bp) cDNA fragment as a hybridization probe for rescreening of the same library, and have thereby recovered a number of additional positive isolates. Of these, three candidates of approximately 900, 1100, and 1300 bp, respectively, were subsequently subcloned and sequenced. Although differing in length, the sequences of these clones were found to be otherwise identical. Moreover, the length of isolate 18B, 1284 bp, corresponded well with that of the single mRNA species (approximately 1300-1350 nucleotides) detected through Northern blot hybridization analysis of rat testis total and poly(A)+RNA. This full- or near full-length cDNA encodes a 315-amino acid protein with a molecular weight of 35,757, in good agreement with the 36,000 estimated molecular weight of heme oxygenase-2. When expressed in Escherichia coli, cDNA encodes a protein that cross-reacts with heme oxygenase-2 antiserum (as assayed by Western immunoblotting) and yields high levels of heme oxygenase activity in bacterial soluble cell extracts. Finally, computer analysis of the heme oxygenase-2 cDNA sequence indicates that the predicted amino acid sequence and hydropathy profile of the heme oxygenase-2 protein exhibit similarity with heme oxygenase-1.
Rotenberg
M O
MO
Department of Biophysics, University of Rochester School of Medicine, New York 14642.
Maines
M D
MD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics* / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006419:Heme Oxygenase (Decyclizing) / Q000235:genetics*; D007527:Isoenzymes / Q000235:genetics*; D006899:Mixed Function Oxygenases / Q000235:genetics*; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011487:Protein Conformation; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D015183:Restriction Mapping
D007527:Isoenzymes; D012333:RNA, Messenger; D004247:DNA; D006899:Mixed Function Oxygenases; D006419:Heme Oxygenase (Decyclizing)