The developmental program controlling sperm formation occurs in multiple stages that sequentially involve mitosis, meiosis, and spermiogenesis. The transcriptional mechanisms regulating these distinct phases are poorly understood. In particular, while a required role for the germ cell transcription factor cyclic AMP response element modulator-during spermiogenesis has recently been demonstrated, the transcriptional mechanisms leading to early haploid cell formation are unknown. The rat and mouse proenkephalin genes are selectively expressed from an alternate, germ cell-specific promoter in meiotic and early haploid cells. In this study, the minimal rat proenkephalin germ line promoter was localized to a 116-bp region encompassing the transcriptional start site region. Further, a proximal 51-bp sequence located in the 5-flanking region is absolutely required for germ line promoter activity. This 51 bp sequence corresponds to a previously characterized binding element (GCP1) that forms cell-specific complexes with rat spermatogenic cell nuclear factors distinct from cyclic AMP response element binding proteins. Further, GCP1 contains novel direct repeat sequences required for factor binding and transgene expression in spermatogenic cells. These repeat elements are highly similar to sequences within the active regions of other male germ line promoters expressed during meiosis. GCP1 may therefore contain transcriptional elements that participate more generally during meiosis in the differentiation of spermatocytes and early haploid spermatids.

In wild-type Escherichia coli, expression of the gal operon is negatively regulated by the Gal repressor and is induced 10- to 15-fold when the repressor is inactivated by an inducer. In strains completely deleted for galR, the gene which encodes the Gal repressor, the operon is derepressed by only 10-fold without an inducer. But this derepression is increased further by threefold during cell growth in the presence of an inducer, D-galactose or D-fucose. This phenomenon of extreme induction in the absence of Gal repressor is termed ultrainduction--a manifestation of further inducibility in a constitutive setup. Construction and characterization of gene and operon fusion strains between galE and lacZ, encoding beta-galactosidase as a reporter gene, show that ultrainduction occurs at the level of transcription and not translation. Transcription of the operon, from both the cyclic AMP-dependent P1 and the cyclic nucleotide-independent P2 promoters, is subject to ultrainduction. The wild-type galR+ gene has an epistatic effect on ultrainducibility: ultrainduction is observed only in cells devoid of Gal repressor protein. Titration experiments show the existence of an ultrainducibility factor that acts like a repressor and functions by binding to DNA segments (operators) to which Gal repressor also binds to repress the operon.

Rat and mouse spermatogenic cells contain a family of 1700-nucleotide (nt) proenkephalin mRNAs that are generated from an alternate, germ cell-specific promoter. This promoter is located approximately 350 base pairs (bp) downstream of the promoter used in somatic cells, within the first intron for the somatic transcript. In a previous study, rat proenkephalin-chloramphenicol acetyltransferase fusion genes containing both promoters were shown to be transcribed selectively from the germ cell promoter and in the correct developmental pattern in spermatogenic cells of transgenic mice. In the present study it was found that spermatogenic cell-specific transgene expression was maintained after deletion of the upstream somatic promoter. This result establishes that the rat proenkephalin germ-line promoter is capable of functioning independently of transcriptional elements associated with the somatic promoter and localizes the requisite spermatogenic cell cis-elements to a 500-bp region encompassing the germ cell initiation sequences. A comprehensive analysis of binding sites for rat spermatogenic cell nuclear factors within this 500-bp region was performed using gel-shift and DNAse I footprinting techniques. Eight distinct binding regions were identified, each of which formed one or more cell-specific complexes with nuclear proteins from rat spermatogenic cells. These results suggest that multiple cis-acting elements may cooperate in the cell-specific and developmental regulation of rat proenkephalin gene transcription during spermatogenesis.

Tn1O insertion in the galS (ultrainduction factor) gene of Escherichia coli allows the gal operon to be constitutively expressed at a very high level, equal to that seen in a AgalR strain in the presence of an inducer. The insertion has been mapped by criss-cross Hfr matings and by marker rescue into Kohara phages at 46 min on the E. coli chromosome.The gal operon of Escherichia coli is regulated both positively and negatively at the level of transcription initiation. There are two gal promoters, P1 and P2; P1 is activated by the cyclic AMP-cyclic AMP receptor protein complex bound in the -40 region, whereas P2 is inhibited by this binding. To isolate a mutation in the gene encoding the second repressor, we started with a strain that was deleted for the galR gene and had a galE-lacZ protein fusion (JT247). (The strains used in this study are shown in Table 1.) In such a strain, ultrainduction was indicated by a threefold increase in ,-galactosidase activity when the inducer was added (10). Colonies of such a strain appeared red on MacConkey lactose plates, even in the absence of an inducer, because of the high level of expression (approximately 500 Miller units; see Table 2). When the P-galactosidase competitive inhibitor thio-ethyl-phenyl-,-D-galactoside (TEPG) (5) of these MC4100 Tcr colonies were pooled, and phage P1 was grown on the bacterial pool. The resulting P1 lysate was used to infect JT247, selecting for tetracycline resistance on Luria broth plates containing 15 ,ug of tetracycline per ml. Tetracycline-resistant colonies were replicaplated to MacConkey lactose plates containing 15 ,ug of tetracycline per ml and 1 mM TEPG. Red colonies were found at a frequency of approximately 1.0%.To ensure that the red phenotype on MacConkey lactosetetracycline-TEPG plates was caused by the ATnJO insertion, phage P1 was used to transfer the ATnJO from six of the mutants into a fresh JT247 strain. In one of the six, there was no linkage (O of 226 tetracycline-resistant colonies) between tetracycline resistance and the MacConkey lactose plate phenotype. In each of the other five, 100% of at least 2,000 tetracycline-resistant colonies were red on the MacConkey lactose-tetracycline-TEPG plates. Only these five were studied further.,-Galactosidase in the mutants was measured and compared with that in the parent strain. Results are shown in Table 2. Two classes of mutants were found: class I, four mutants that in the absence of an inducer had a high level of gal-lacZ expression which increased further upon addition of an inducer (represented by strain AG708); and class II, one mutant that in absence of an inducer had a high level of expression of gal-lacZ which did not increase upon addition of an inducer (strain AG701). The phenotype of the second class was what we expected from a mutation in a gene encoding the proposed second gal repressor. Expression was at a high level regardless of whether an inducer was present. We call the gene into which the ATnJO was inserted galS. The mutation in AG708 is probably not allelic...