Testis

Mouse Testin: Complementary DNA Cloning, Genomic Organization, and Characterization of Its Proximal Promoter Region

Abstract

Testin is a secretory protein initially identified from rat Sertoli cell-enriched cultures and has been suggested to be a sensitive marker to monitor the integrity of Sertoli-germ cell junctions. However, neither the expression of testin gene in other species nor the molecular mechanisms that govern its transcription is known. To address these issues, we cloned and characterized the mouse testin gene. A full-length mouse testin cDNA encoding a polypeptide of 333 amino acid residues was isolated by library screening. Sequence analysis revealed that mouse testin shared 90.1%, 58.9%, 62.2%, and 64.6% identity with rat testin and cathepsin L of mouse, rat, and human, respectively, at the amino acid level. Reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis demonstrated that mouse testin transcripts were predominantly expressed in the gonads. The mouse testin gene spans over 21 kilobases (kb) and contains eight exons interrupted by seven introns. Primer extension analysis and 5'-Rapid Amplification of cDNA Ends (5'-RACE) identified a major transcription start site located 134 base pairs (bp) upstream from the translation initiation codon. Analysis of a 2.3 kb mouse testin 5;-flanking region revealed that it lacked TATA and CAAT boxes, and the region was not GC-rich. By the use of deletion analysis, in vitro DNase I footprinting and site-directed mutagenesis, we have identified within the proximal promoter region three closely-spaced putative binding sites for GATA, sex-determining factor (SRY), and steroidogenic factor-1 (SF-1) that are important for testin gene transcription in mouse Sertoli MSC-1 cells. Interestingly, these cis-acting elements are also present in the conserved Mullerian inhibiting substance (MIS) proximal promoters, raising a possibility that the transcriptions of testin and MIS genes are controlled by similar mechanisms.