Immunoglobulin alternative RNA processing is regulated during B-cell maturation and requires balanced efficiencies of the competing splice (m) and cleavage-polyadenylation (s) reactions. When we deleted sequences 50 to 200 nucleotides beyond the s poly(A) site, the s/m mRNA ratio decreased three-to eightfold in B, plasma, and nonlymphoid cells. The activity could not be localized to a smaller fragment but did function in heterologous contexts. Our data suggest that this region contains an RNA polymerase II pause site that enhances the use of the s poly(A) site. First, known pause sites replaced the activity of the deleted fragment. Second, the fragment, when placed between tandem poly(A) sites, enhanced the use of the upstream poly(A) site. Finally, nuclear run-ons detected an increase in RNA polymerase loading just downstream from the s poly(A) site, even when the poly(A) site was inactivated. When this fragment and another pause site were inserted 1 kb downstream from the s poly(A) site, they no longer affected the mRNA expression ratio, suggesting that pause sites affect poly(A) site use over a limited distance. Fragments from the immunoglobulin A gene were also found to have RNA polymerase pause site activity.Elements that modulate gene expression by altering RNA polymerase elongation (reviewed in references 9, 35, 43, and 49) have been identified near the 5Ј end of genes (e.g., myc and human immunodeficiency virus) (18,20,26,27), within the coding region of genes (e.g., histone H3.3 and apolipoprotein A-I) (22,37,46), and downstream from some (3, 14), but not all (51), cleavage-polyadenylation [poly(A)] sites. The elements found downstream from the ␣ globin (14) and complement C2 (3) poly(A) sites were shown to contribute to transcriptional termination. The ␣ globin element is contained within a 92-bp fragment that is located about 300 nucleotides (nt) downstream from the ␣ globin poly(A) site (14). The C2 element is a 156-bp fragment that begins just downstream from the C2 poly(A) site; despite its close proximity, it is not required for efficient use of the C2 poly(A) site (3). These elements were called RNA polymerase pause sites because they appeared to have a kinetic affect on RNA polymerase II transcription (3, 14); when placed between two competing reactions, their effect on gene expression was consistent with these elements causing RNA polymerase to pause or slow its elongation rate. This was true when they were placed between tandem poly(A) sites (3,14), between tandem promoters (13), and between a regulated exon and its intronic regulatory region (38). In addition, in nuclear run-on assays, which measure RNA polymerase loading along a transcription unit, an increase in RNA polymerase loading was seen over these elements, again suggesting that RNA polymerase was being slowed or temporarily paused, so there was a higher probability of finding RNA polymerase molecules over those regions (3,14). More recently, the activity of these pause sites and their effect on upstream poly(A) sites was examined in...
Sensory neuron development and differentiation is dependent on a family of growth factors known as neurotrophins. Neurotrophins modulate neuron development via trk tyrosine kinase receptor proteins trkA, trkB and trkC. To determine how elevated levels of a target-derived neurotrophin might affect neuronal differentiation, we analysed trk expression in the trigeminal ganglion of transgenic mice that overexpressed nerve growth factor (NGF) in the skin. Increased levels of NGF caused a five-fold increase in neurons expressing trkA mRNA and a two-fold increase in neurons expressing trkC. In control mice, cell size distributions of neuronal subpopulations expressing each trk mRNA showed the three subpopulations distributed over a narrow, overlapping range. In contrast, cell size distribution in NGF-transgenic mice was significantly divergent due in large part to hypertrophy of trkA neurons and, to a lesser extent, trkC neurons. In addition, we examined neurons that bound the isolectin B4 from Bandeiraea simplicifolia (BS-IB4) because most of these neurons do not express any trk receptor in the adult. There was a significant increase in the size of BS-IB4-positive neurons in transgenic mice; however, there was no increase in their number. These studies indicate that an increased level of target-derived NGF affects the development of sensory neurons that in the adult express trkA or trkC, as well as neurons that do not express trk receptors.
Abstract. To examine the role of keratin intermediate filament proteins in cell structure and function, transgenie mice were isolated that express a modified form of the human K14 keratin protein in liver hepatoeytes. A modified K14 eDNA (K14.P) sequence was linked downstream of the mouse transthyretin (TTR) gene promoter and enhancer elements to achieve targeted expression in hepatocytes. Hepatoeytes expressing high levels of the transgene were found to have abnormal keratin filament networks as detected by indirect immunofluorescenoe using an antibody specific for the transgene product. Light and electron microscopic level histological analysis of isolated liver tissue showed in many eases degenerative changes that included inflammatory infiltration, ballooning degeneration, an increase in fat containing vacuoles, and glycogen accumulation. These changes were most evident in older mice over four months of age. No indication of typical Mallory body structures were identified at either the light or electron microscopic level. To evaluate secretory function in transgenic livers, bile acid secretion rates were measured in isolated perfused liver and found to be approximately twofold lower than aged-matched controls. These findings indicate that expression of an abnormal keratin in liver epithelial cells in the in vivo setting can alter the structure and function of a tissue and suggest a role of the keratin network in cellular secretion.
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