POSTER NO: 210

Isolation and Charateration of Two Human TCP11 Gene Isoforms of Resulted from Complicated Splicing

Yongxin Ma, Sizhong Zhang
Department of Medical Genetics, West China Hospital, Sichuan University, Renminnanlu No.17, Section 3, Chengdu, 610041, P.R.China

The mouse t-complex is known to harbour a series of genes affecting male fertility. As one of t-complex genes, Tcp-11 is only expressed during male germ cell development and encodes the receptor for fertilization promoting peptide (FPP),which stimulates the mouse and human sperm capacitation.

By using subtractive hybridization between the cDNA pools of fertile adult and fetal testes followed by rapid amplification of cDNA ends (RACE), we have identified the first isoform of the human homologue of Tcp11 and deposited it into GenBank/EMBL/DDBJ under Accession No.AF269223. This 1722bp sequence is highly homologous to a large portion of the mouse Tcp11 gene. The processed mature mRNA encodes a 441 amino acid protein that is homologous to the mouse 566 amino acid protein after the first 142 amino acids.

Recently, during the homology analysis of the sequence AF269223 with BLAST, some ESTs whose 5'-ends were different from AF269223 but the 3'- ends were identical to AF269223 were found. These results suggest that there may exist other transcripts distinct from AF269223 in their 5'-sequence. From these transcripts we obtained a novel isoform of human TCP11 by PCRs with two pairs of primers. This sequence was deposited in GenBank/EMBL/DDBJ under Accession No. AY069943. The novel transcript encodes a 503 amino acid protein. Its 3'-1466bp of the 1746bp sequence is completely identical to AF269223 and spans 6 exons from the 3'end. So the C-terminal 425 amino acid sequences of the two proteins should be identical. But the splicing of the sequences before the 6th exons from the 3'-end of both transcripts were different and complicated.

The gene has been mapped to human chromosome band 6p21 by fluorescence in situ hybridization. This gene spans about 22kb genomic DNA sequence. Northern blot and RT-PCR analyses of RNA extracted from human tissues revealed that both the transcripts were expressed only in fertile adult testes, but not in testes of azoospermic patient and fetus, nor in other human tissues. It is interesting to study the function of the two isoforms of the same gene in human spermatogenesis and fertility.