We present evidence for the existence of prolactin upstream factor I (PUF-I) in rat pituitary-derived cells and demonstrate its interaction with a symmetrical DNA element located in the 5' flanking region of the gene. An in vitro expression system developed from pituitary-derived Gil3 cells was used to determine that 420 base pairs (bp) of 5' flanking DNA was sufficient for cell-specific, accurate, and efficient RNA polymerase II transcription of the rat prolactin gene. Reconstitution of in vitro transcription with pituitary and nonpituitary nuclear extracts suggested that the presence of GH3 cell-specific factors mediated the activation of prolactin gene expression. We also demonstrated that a functionally stable transcription complex assembled on the prolactin promoter. Using DNase I protection procedures, we have identified the DNA-protein binding area in the prolactin 5' flanking region. Gil3 nuclear extracts contain a cell-specific protein (PUF-I) that binds to a 28.bp region (-63 to -36) which contains an 18-bp imperfect palindrome (-63 to -46). The role that the interaction between PUF-I and the imperfect palindrome plays in in vitro pituitary-specific prolactin gene expression is discussed.The prolactin gene is expressed primarily in mammalian pituitary lactotrophs and is regulated by inhibitory and stimulatory hormones from the brain and gonads (6) plus several other classes of factors including Ca2+ (12), phorbol esters (22), and cyclic AMP (16). Previous studies have suggested that most of these factors modulate prolactin production at the level of gene transcription (2,15,16,19,21). Regulation of gene activity is presumably mediated by the interaction of cis-acting promoter elements with transacting nuclear protein transcription factors (8). The 5' flanking prolactin DNA contains homologous sequences for several eucaryotic cis control elements including two TATA boxes at positions -28 to -23 and -142 to -136 and a CAAAT sequence from -192 to -187. In addition, there is an interesting A+T-rich 18-base-pair (bp) imperfect symmetrical sequence from -63 to -46 (4, 17).An investigation of the prolactin cis-acting elements by Nelson et al. (20), using gene transfer methods, suggested the presence of two discrete, separable sequences which act in a position and orientation independent manner to enhance prolactin gene transcription. It was proposed that these two regions (from -1768 to -1712 and from -1653 to -1531) are cell-specific enhancer elements which mediate the activation of these genes in lactotrophs and somatotrophs.We have used a different approach to study the interaction of cis-acting DNA sequences and trans-acting elements leading to an activated rat prolactin gene. Cell-free nuclear extracts prepared from a rat pituitary-derived cell line, GH3, and nonpituitary cells were used to study the cell-specific in vitro transcription and DNA protein-binding interactions of the prolactin gene. We combined transcriptional and binding assays to identify DNA sequences upstream from the prolactin gene whic...

DNA sequence-specific chromatography was used to purify prolactin upstream factor I (PUF-I) approximately 10,000- to 20,000-fold from rat GH3 cells. The purified transcription factor reconstituted enhanced pituitary-specific prolactin RNA synthesis in nonpituitary in vitro transcription assays. In vitro mutagenesis demonstrated that the capacity to stimulate prolactin gene transcription was directly correlated with PUF-I binding to an A+T-rich region located from -63 to -36 in the prolactin 5'-flanking DNA. We propose that PUF-I is a critical modulator of transcriptional activity in pituitary cells and has a central role in the stimulation of prolactin gene transcription in the mammalian pituitary lactotroph.

Within the promoter region of the rat prolactin gene lies a TA-rich imperfect palindrome. The possible functions of the 18-base-pair symmetrical sequence were investigated by using an in vitro transcription system. Prolactin templates with and without the palindrome were transcriptionally assayed in both pituitary and nonpituitary extracts. Our results indicated that the palindromic sequence has at least two functions in the regulation of prolactin transcription.Enhancer elements are typically located a considerable distance from the transcriptional initiation site at position + 1 and have been shown to stimulate transcription in a tissuespecific manner in many gene systems (12,18). In addition, several recent studies have demonstrated that cis-acting elements within the proximal 5' region of some genes are also involved in cell-specific regulation (1,7,10,20). In the rat prolactin gene, an enhancer from positions -1850 to -1530 and a region of the promoter between positions -422 and -36 have been shown to augment prolactin transcription in pituitary-derived cells but not in nonpituitary-derived cells (14).Within the segment of the rat prolactin promoter from positions -422 to -36 lies an 18-base-pair (bp) imperfect palindrome, TGATTATATATATATTCA (13). Since sequences with complete or limited dyad symmetry are found frequently within regulatory elements of both procaryotic (15) and eucaryotic genes (2,8,9,11,16,17,19), it is plausible to suspect that the palindrome (positions -63 to -46) might be involved in the cell-specific regulation of prolactin transcription.This symmetrical sequence also contains five sequential TA repeats. The nucleotide composition of the palindrome is of particular interest since it constitutes a TATA homology (M. L. Goldberg, Ph.D. thesis, Stanford University, Stanford, Calif., 1979). This has raised the possibility of a second TATA element upstream of the proximal TATA box (positions -28 to -23).In the present study, the possible functions of the palindrome were investigated. Prolactin templates with and without the palindrome were assayed in pituitary-and nonpituitary-derived nuclear extracts to determine whether the palindrome is involved in the cell-specific regulation of prolactin transcription. Furthermore, since the palindrome TA repeats are homologous to the TATA consensus se- quence, the possibility of an mRNA initiation site 20 to 30 bp downstream of the palindrome was also explored.Prolactin transcription in vivo. The in vivo sites of prolactin transcriptional initiation were determined by primer extension analysis of total RNA extracted from GH3 cells (Fig. 1). These cells, which were derived from a rat pituitary tumor, constitutively produce high levels of prolactin. Approximately 10 ,ug of GH3 RNA was isolated by a modified guanidine hydrochloride procedure (5), and the RNA was assayed by primer extension analysis. An oligonucleotide primer complementary to prolactin RNA was used to detect two sites of transcriptional initiation, a major one at position + 1 and a minor RN...

The cell-type-specific transcription of the prolactin gene in vitro is mediated through the interaction of prolactin upstream factor I (PUF-I) with a 28 basepair region of the gene promoter (-63 to -36) which contains an 18 bp A+T-rich imperfect palindrome (-63 to -46). Base substitutions were introduced into 16 of the 18 palindromic residues by targeted saturation mutagenesis. The GH3 binding and in vitro transcription assays of the mutated promoters showed that base substitutions within the 5'-ATATTCA-3' sequence located at -52 to -46 were detrimental to PUF-I binding and its cell-type-specific transcriptional enhancement activity. Transcription assays of the mutated promoters performed with several nonpituitary-derived extracts demonstrated that a distal TATA box located from -59 to -53 promotes initiation at -27. Thus, the cell-type-specific cis-acting element required by PUF-I for DNA recognition is immediately adjacent to a general TATA sequence. Base substitutions that decreased +1 transcription and PUF-I binding concomitantly increased -27 initiation of RNA in vitro. We suggest that PUF-I binding in pituitary cells potentiates +1 transcription and represses alternative TATA box activity for initiation events occurring at -27. This is the first known report of a eukaryotic DNA binding protein that has both an activator and repressor activity for a single transcription unit.

Removal of up to 50% of the sialic acid from aggregates of 7-day chick embryo heart cells during incubation in a highly purified preparation of neuraminidase resulted in hyperpolarization of the maximum diastolic potential, reduction in the slope of diastolic depolarization leading to slowing of beating, negative shifts of threshold potential and the voltage at which upstroke velocity was maximal, and an initial increase in the action potential overshoot. These effects were opposite to those induced by phospholipase C, a possible contaminant. The modification of electrical properties by neuraminidase is suggested to result from an enhanced influx of calcium ions that might "screen" or bind specifically to internal negative fixed charges and thereby shift the voltage dependence of conductance and kinetic parameters to more negative potentials. This hypothesis is supported by the results above and by greater uptake of 45Ca following release of sialic acid, augmentation of enzyme-induced changes in elevated Ca2+, and the similarity of effects produced by A23187, and inophore which also increased 45Ca uptake. However, other mechanisms cannot be ruled out at the present time.