Abstract
The GP1G gene of the guinea pig codes for three of the four abundant seminal vesicle secretory proteins produced in this species. This gene is expressed at highest efficiency in the seminal vesicle (SV) from a promoter that contains a canonical TATA box and CCAAT box. However, GP1G gene transcripts and proteins have also been identified in other tissues. To investigate the structure of GP1G transcripts produced in the testis, cDNA clones were isolated by screening a testis library. Three unique cDNAs (TSM1-3) were isolated. Each of these clones contained a 3'-untranslated region (UTR) and coding region identical to that of the seminal vesicle transcript. However, the 5'-UTRs of the testis transcripts were significantly longer than that found on the SV mRNA (416-646 nucleotides compared with only 23 nucleotides for the SV). Each of these alternatively spliced 5'-UTRs incorporated the SV promoter elements into transcribed sequence, and each contained multiple upstream AUG codons predicted to abolish translation of the major open reading frame. Nevertheless, each of the testis transcripts was capable of directing the synthesis of GP1G-related proteins in vitro. Analysis of the translation products suggests that the extended 5'-UTR of the testis transcripts regulate both the choice of translation start site and the efficiency of translation in this system. Western blot analysis of testis proteins revealed that the protein products of GP1G are also synthesized by the testis in vivo.
Highlights
The seminal vesicle secretory proteins are thought to contribute to maintaining the integrity and transport of spermatozoa [1]
Using the first 175 bp of testis-specific message 1 (TSM1) as a probe, an additional screening of the library yielded TSM3, which is identical to the other GP1G transcripts in the 3Ј-untranslated region (UTR) and coding regions, but had an additional 77-bp internal deletion immediately upstream of the 153-bp deletion found in the 5Ј-untranslated region (5Ј-UTR) of TSM2 (Fig. 2)
The data presented here demonstrate that the GP1G gene is expressed in the testis as well as the seminal vesicle, and highlight new regulatory mechanisms that are involved in the production of proteins from this locus
Summary
Library Screening—A guinea pig testis cDNA library (custom made by Stratagene in Lambda-Zap II) was screened with either a 32P-labeled random primed 1400-bp GP1 probe [3] or a 175-bp 5Ј-end fragment from TSM1. Northern Blot Analysis—Total RNA was isolated from guinea pig testis or seminal vesicle [17]. Novel SVP-1/-3/-4 Transcripts in Guinea Pig Testis mM Tris, pH 6.8, 20% glycerol, 4.6% SDS, 10% -mercaptoethanol, 0.001% bromphenol blue, and boiled for 5 min. Amplified DNA was cloned into pCR2.1 using TA cloning kit (Invitrogen; Cat. K2000 – 01) and sequenced: 35299, 5Ј-CAGCAGAGATCATAGAG-3Ј (complementary to nts 284 – 300 shown on Fig. 2); 35191, 5Ј-ATCACAAAGTTTCCAGTG-3Ј (complementary to nts 239 –243, 397– 409); 35503, 5Ј-ACCAATCTCTCTGGAAC-3Ј (complementary to nts 488 –504); 35122, 5Ј-GATCACTAGTCCTACGCAGTGCCTCTTC-3Ј; and 35479, 5Ј-GATCACTAGTAGGCATATGATGTCAGAGAC-3Ј. The resulting constructs contain full-length TSM1–3 cloned into EcoRI and XhoI sites in pcDNA3. In Vitro Transcription/Translation—TSM1–3 and GP1 cDNA clones in pcDNA3 were linearized with XhoI. The gel was fixed, dried, and analyzed using a Molecular Dynamics STORM 840 PhosphorImager
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