August 1996
Volume 37, Issue 9
Free
Articles  |   August 1996
Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein.
Author Affiliations
  • N S Bora
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • P S Bora
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • M T Tandhasetti
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • T P Cirrito
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • H J Kaplan
    Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
Investigative Ophthalmology & Visual Science August 1996, Vol.37, 1877-1883. doi:
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      N S Bora, P S Bora, M T Tandhasetti, T P Cirrito, H J Kaplan; Molecular cloning, sequencing, and expression of the 36 kDa protein present in pars planitis. Sequence homology with yeast nucleopore complex protein.. Invest. Ophthalmol. Vis. Sci. 1996;37(9):1877-1883.

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Abstract

PURPOSE: 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.

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