Cloning and sequencing of a cDNA encoding for a protein specifically expressed in human testis, ovary and placenta.
Reproductive cell/tissue-specific antigens are attractive candidates for the development of a contraceptive vaccine. Using the differential display technology, a human cDNA fragment of 322-bp, designated as T17, was identified showing specific expression in the human testis. The T17 cDNA fragment was used as a probe to screen the human testis cDNA-lambda gt10 library. After screening, one positive clone of approximately 1.1 kb having T17 nucleotide (nt) sequence was obtained. The 5' and 3' termini of this cDNA clone were extended by using 5' and 3' RACE procedures that yielded a full-length cDNA, designated as the TOP gene. The TOP cDNA is 1,480-bp long and has an ORF of 463 aa with the first ATG Met start codon at nucleotide (nt) 64 and the stop codon TGA at nt 1452. The translated protein has a calculated molecular mass of 49.3 kD with isoelectric point of 12.17. The deduced amino acid (aa) has one potential N-glycosylation site, and several phosphorylation and myristoylation sites. Hydropathy plot generated from the deduced aa sequence showed it to be a surface protein. Comprehensive computer search in the database did not reveal any homology to any existing sequences both at the nt and aa levels. The TOP cDNA was found to be completely localized on the human chromosome 16 at the nt position 507833-506354, with the TOP ORF at the nt position 507770-506354. Northern blot analysis using three human Northern blots, indicated the specific expression of TOP gene in the human testis, ovary and placenta. RT-PCR-Southern blot analysis also confirmed tissue-specific expression of the TOP gene. The TOP cDNA may help us to gain insight into transcriptional control of the differentially expressed reproductive tissue-specific genes. It may also find clinical applications in the development of a contraceptive vaccine, and specific diagnosis and treatment of infertility in humans.
Naz
Rajesh K
RK
Division of Research, Department of Obstetrics and Gynecology, Health education Building, Rm. 211, Medical College of Ohio, 3055 Arlington Avenue, Toledo, OH 43614-5806, USA. Rnaz@mco.edu
Zhu
Xiaolong
X
Rajesh
Changanamkandath
C
D015152:Blotting, Northern; D015139:Blotting, Southern; D003001:Cloning, Molecular; D003062:Codon; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D020869:Gene Expression Profiling; D015723:Gene Library; D006801:Humans; D008297:Male; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D016366:Open Reading Frames; D010053:Ovary / Q000378:metabolism*; D010766:Phosphorylation; D010920:Placenta / Q000378:metabolism*; D062085:RNA, Long Noncoding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013737:Testis / Q000378:metabolism*; D014018:Tissue Distribution
D003062:Codon; D018076:DNA, Complementary; C509980:LINC00273 lncRNA, human; D008562:Membrane Glycoproteins; D062085:RNA, Long Noncoding