Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein.
OBJECTIVE
Patients with active pars planitis have increased levels of a 36 kDa protein (p-36) in their circulation. The current studies were undertaken to determine the primary structure of this protein.


METHODS
A degenerate oligonucleotide probe based on the amino terminal sequence of p-36 was used to identify a clone from a human spleen cDNA library. The cDNA insert was subcloned into the EcoR1 site of pUC-19, and both strands were sequenced. Southern blot analysis was used to study the genomic hybridization pattern. p-36 cDNA was subcloned in a pSG5 expression vector, and the construct was used to transfect COS-7 cells.


RESULTS
The cDNA sequence contained an open reading frame of 966 base pairs encoding a protein of 322 amino acids, an untranslated region of 322 base pairs, and 2693 base pairs at the 5' and 3' ends, respectively. The deduced amino acid sequence showed 96.8% identity with the carboxy-terminal region of a yeast nucleopore complex protein, nup 100. Southern blot analysis of human genomic DNA revealed a simple hybridization pattern. Transfection of p-36 cDNA in COS-7 cells resulted in the presence of p-36 mRNA and expression of protein.


CONCLUSIONS
The 36 kDa protein (p-36) detected at increased levels in the blood of patients with active pars planitis was cloned from a human spleen cDNA library. Its deduced amino acid sequence is homologous with the carboxy-terminal region of a nucleopore complex protein. Thus, we refer to this protein as nup36.
Bora
N S
NS
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
Bora
P S
PS
Tandhasetti
M T
MT
Cirrito
T P
TP
Kaplan
H J
HJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001798:Blood Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D015139:Blotting, Southern; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004247:DNA / Q000097:blood; D018076:DNA, Complementary; D006801:Humans; D007962:Leukocytes / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins / Q000096:biosynthesis* / Q000097:blood / Q000737:chemistry; D015345:Oligonucleotide Probes; D016366:Open Reading Frames; D015868:Pars Planitis / Q000097:blood*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013154:Spleen / Q000378:metabolism*; D014162:Transfection
D001798:Blood Proteins; D018076:DNA, Complementary; D028861:Nuclear Pore Complex Proteins; D009687:Nuclear Proteins; D015345:Oligonucleotide Probes; D011994:Recombinant Proteins; D004247:DNA