[The characterization of a novel testis-specific nucleoporin gene].
OBJECTIVE
To investigate the structure and function of testis-specific gene and spermatogenesis in human.


METHODS
Screening cDNA expression library, 5' rapid amplification of cDNA ends, Northern blot and fluorescent in situ hybridization(FISH) were used. Gene expressing, purified of expressed protein by affinity chromatography and SDS-PAGE as well as phosphorylation of expressed protein in vitro by PKC and p34cdc2 were observed.


RESULTS
A cDNA designated as BS-63 was isolated and found to consist of 2,209 bp with an open reading frame of 2,100 bp and assigned the accession number U64675 by GenBank. The deduced polypeptide consisted of 700 amino acid residues containing XFXFG or FG motifs that were characteristic of nuclear pore complex (NPC) protein and acted as potential binding sites for Ran. The N-terminal region had high homology with Ran BP2/Nup 358, a nucleoporin component, showing that BS-63 was a member of the NPC family. Northern blot analysis of mRNA prepared from various human tissues showed that BS-63 gene was transcribed in two forms: 6.0 and 8.5 kb. The 8.5 kb transcript was present in low amounts in several somatic tissues; whereas the 6.0 kb transcript was expressed only in testis. Analysis by FISH method mapped the BS-63 gene in 2q11.2-12. A protein band with an estimated Mr of 80,000 was detected with E. coli BL21 (DE3) transfected with recombinant plasmid pET30a (+)-BS-63. In vitro phosphorylation test indicated the BS-63 recombinant protein could be phosphorylated by PKC and p34cdc2.


CONCLUSIONS
The study was the first demonstration that the BS-63 gene encoding a nucleoporin-related protein with Ran binding sites was expressed in germ cells of human testis.
Gao
Y
Y
National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, CAMS and PUMC, Beijing 100005.
Zhu
H
H
Cao
D
D
Miao
S
S
Wang
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D002889:Chromosomes, Human, Pair 2; D018076:DNA, Complementary / Q000235:genetics*; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D008297:Male; D008969:Molecular Sequence Data; D028861:Nuclear Pore Complex Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D013737:Testis / Q000737:chemistry*
D018076:DNA, Complementary; D028861:Nuclear Pore Complex Proteins; D012333:RNA, Messenger