Prolactin upstream factor I mediates cell-specific transcription.

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.

Reconstitution of cell-type-specific transcription of the rat prolactin gene in vitro.

We present evidence for the existence of prolactin upstream factor 1 (PUF-1) 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 GH3 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. GH3 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.

Dual functions of a cis-acting element within the rat prolactin gene promoter.

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.

Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1.

PURPOSE: To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to rat platelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs. METHODS: Magnetic beads were coated with anti-rat PECAM-1 antibodies. Retinas were obtained from Lewis rat eyes. After the retinas were digested with collagenase, they were incubated with the antibody-coated beads with agitation. RECs that stuck to the beads were collected with a magnetic particle concentrator and cultured in fibronectin coated wells. The characteristics of the RECs were examined by immunohistochemical study utilizing von Willebrand's Factor, acetylated low-density lipoprotein uptake and transmission electron microscopy. RESULTS: The cells isolated using the PECAM-1-coated magnetic-bead technique formed a contact-inhibited cobblestone monolayer that stained positive for von Willebrand's Factor. These cells revealed low-density lipoprotein uptake. Ultrastructurally, the isolated cells exhibited pinocytic vesicles and a high density of intercellular junctions without fenestrations. CONCLUSION: These results indicate that the isolated cells were vascular endothelial cells showing both morphologic and functional characteristics of retinal vascular endothelium. The magnetic-bead technique was useful for isolating high purity RECs that can be cultured to study the physiological, immunological and biochemical role of the endothelium in various ocular diseases.

DNA recognition element required for PUF-I mediated cell-type-specific transcription of the rat prolactin gene.

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.

Sialic acid: regulation of electrogenesis in cultured heart cells.

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.