Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system.

Abstract

Transcriptionally active nuclear extracts were prepared from mouse testes to study the transcription of the testis-specific mouse protamine 2 (Prm-2) gene in vitro. The testicular system is unique among mammalian in vitro transcription systems in regard to its temperature optimum. In extracts made from prepuberal testes, the temperature optimum for in vitro transcription of Prm-2 is 30°C, similar to somatic in vitro systems. However, in adult testis extracts, the optimum temperature for Prm-2 transcription is 20°C. The different temperature optima seen in vitro for prepuberal and adult testes extracts parallels in vivo physiological temperature sensitivities of the differentiating male germ cells. The testis system also differs from other in vitro transcription systems in its divalent metal cation and ionic strength requirements for optimal transcription. The mouse Prm-2 gene is maximally transcribed at a MgCI2 concentration of 3-5 mM and over a KCI concentration range of 40-100 mM. By using the testis in vitro transcription system to study the Prm-2 gene by deletion analysis, we have determined that positive promotion for the gene lies within the region -170 to -82 from the start of transcription. This region contains a putative Sp-l binding site. Additional upstream sequences appear to repress Prm-2 transcription in a heterologous transcription system.

Factors involved in regulation of the RT7 promoter in a male germ cell-derived in vitro transcription system.

“…Our finding that RT7 promoter activity is at least as high in liver nuclear extracts as in ST nuclear extracts indicates that the complete mechanisms that underlie RT7 tissue-specific gene transcription are not reproduced in vitro. These findings are similar to those reported for the mP2 promoter (16); an mP2 fragment that is necessary and sufficient to give maximal transcription in total testis extracts is equally active in HeLa cell extracts (16). With these caveats in mind, the following conclusions can be drawn concerning the regulation of RT7 promoter activity in vitro by trans-acting factors.…”

“…Seminiferous tubules (STs) rather than total testis constitute a preferred source of nuclear extract in the study of male germ cell-specific promoters; in contrast to STs that contain mostly male germ cells (>95%) and a small number of Sertoli cells but no other somatic cells, total testis contains a large number of cells other than male germ cells (such as Leydig cells, fibroblasts, myoid cells, etc.). In the case of transcriptional repressor mechanisms similar to those reported for the mP2 promoter (16), these may obscure the results. Different RT7 promoter fragments were tested for activity in vitro using the male germ cell system as well as a liver nuclear extract.…”

“…Thus our data suggest that one or more of these protein binding sites may exert a small negative effect on RT7 promoter activity, whereas the effect of the other sites may not be detectable in vitro. Interestingly, it has been reported by others that the effect of NF-1 sites [e.g., in the albumin promoter (6,7) and the mP2 promoter (16)] cannot be demonstrated in vitro.…”

“…For comparison, we also tested the RT7 promoter constructs in liver nuclear extracts. The use of homologous and heterologous extracts helps distinguish tissue-specific and ubiquitous transcription factors involved in promoter regulation (6,16,21,22).…”

“…3B). In addition, regions with homology to the B and D elements (14, 15), which were identified by virtue of their sequence conservation in male germ cell-specific promoters mP1, mP2, and PGK-2, are present; Bunick et al (16) concluded that the B and D elements are involved in positive and negative transcription regulation of the mP2 promoter, respectively. Thus, given the extensive homology of RT7 promoter sequences with these transcription factor regulatory elements it was of interest to determine their role, if any, in RT7 promoter regulation.…”