Identification of a splice variant mRNA of p40phox, an NADPH oxidase component of phagocytes.
Northern blot analysis using p40phox cDNA probe revealed that two sizes of p40phox mRNAs were expressed in human promyelocytic HL-60 and bone marrow cells. To characterize these mRNAs, we performed reverse transcription using total RNA from HL-60 cells, and amplified the coding region of p40phox by polymerase chain reaction with oligonucleotide primers. Two cDNA fragments with different sizes were isolated. One was identical to a known p40phox cDNA (1054 bp) which encoded a protein of 339 residues (39,031 Da) with a calculated pI of 6.5. The other cDNA (1299 bp) contained an additional 245 bp intron 8 sequence in the open reading frame and encoded a protein of 348 residues (39,000 Da) with a calculated pI of 9.3. N-terminal 253 residues were identical between p40phox and the variant protein, whereas C-terminal 254-348 residues of the variant protein shared low homology with p40phox. Interestingly, the variant protein lacked PC (Phox and Cdc24p) motif of p40phox, which is assumed to be important for the interaction with p67phox. In addition, Western blot analysis revealed that the variant protein was not detected in HL-60 cells and neutrophils. Together, these observations suggest that alternatively spliced variant mRNA of p40phox is expressed, but its protein is hardly present in myeloid cells.
Hasebe
T
T
Department of Biochemistry, Juntendo University, School of Medicine, Tokyo, Japan.
Someya
A
A
Nagaoka
I
I
D017398:Alternative Splicing; D000595:Amino Acid Sequence; D001483:Base Sequence; D001854:Bone Marrow Cells / Q000378:metabolism; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D018922:HL-60 Cells; D006801:Humans; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000235:genetics*; D010586:Phagocytes / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D010750:Phosphoproteins; D012333:RNA, Messenger; C085072:neutrophil cytosol factor 40K; D019255:NADPH Oxidases