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a Department of Cell Biology and the Center for Recombinant Gamete Contraceptive Vaccinogens, University of Virginia School of Medicine, Charlottesville, Virginia 22908
b Ludwig Institute for Cancer Research, London W1W 7BS, United Kingdom
c BioQual, Inc., Rockville, Maryland 20850
The human flagellar protein tektin B1 (h-tekB1) in human sperm was cloned, and its sequence and subcellular location were determined. Human sperm proteins were separated by 2-dimensional electrophoresis, and a resolved protein spot of 54 kDa with an isoelectric point (pI) of 5.3 was removed from the gel, trypsinized, and microsequenced by tandem mass spectrometry. The resulting peptides did not match any protein in the (then current) protein databases. Degenerate oligonucleotides based on the microsequences were used with a polymerase chain reaction to amplify a partial cDNA clone from human testis poly(A)+ mRNA, and subsequently a full-length 1.5-kilobase (kb) clone (GenBank AF054910) was obtained from a testis cDNA library. The open reading frame encoded a 430-amino acid protein with 47% homology to the sea urchin tektin B1. Hybridization of labeled h-tekB1 cDNA to a multiple-tissue Northern blot demonstrated a transcript of 1.7 kb in human testis, and a multiple tissue dot-blot demonstrated high levels of expression in testis, trachea, and lung, intermediate levels in fetal brain and appendix, and low levels in ovary, pituitary, and fetal kidney. Rat polyclonal serum generated against a recombinant h-tekB1 demonstrated 3 h-tekB1 isoforms of pI 5.25, 5.5, and 5.35 at 53.5 kDa on a 2-dimensional Western blot of human sperm proteins. Immunofluorescent studies localized h-tekB1 to the principal piece of human sperm, but the endpiece was unstained.
1 This work was supported by NIH U54 HD29099 and a grant from the Andrew W. Mellon Foundation. Note: The clone sequence from this study was assigned GenBank accession number AF054910. During the preparation of this paper, a human clone (accession AB033823) was submitted to GenBank on 12 January 2000 by Iguchi et al. AB033823 differs from our clone (submitted on 2 April 1998) by 66 of the 430 amino acids.
2 Correspondence: John C. Herr, Department of Cell Biology, Box 439 Jordan Hall, University of Virginia, Charlottesville, VA 22908. FAX: 804 982 3912; jch7k{at}virginia.edu
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