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At least 19 recordsLinked to original sources
PubMed1992

Differential phosphorylation and localization of the transcription factor UBF in vivo in response to serum deprivation. In vitro dephosphorylation of UBF reduces its transactivation properties.

We have analyzed the expression, phosphorylation, and localization of the ribosomal DNA transcription factors UBF1 and UBF2 in Chinese hamster ovary cells in response to serum deprivation. In vivo labeling experiments demonstrate that UBF1 and UBF2 are phosphoproteins. Phosphoamino acid analysis of the in vivo labeled proteins demonstrate that UBF is phosphorylated on serine residues. Following serum deprivation there is no alteration in the cellular levels of UBF1 and UBF2 as determined by Western blotting, but there is an 80% reduction in the level of phosphorylation of UBF compared with logarithmically growing cells. Following serum deprivation there is a redistribution of UBF between the nucleolus, the nucleus, and the cytoplasm. Phosphatase-treated UBF demonstrated a reduced ability to rescue transcription by RNA polymerase I from the rDNA spacer promoter in vitro. These findings suggest that phosphorylation of UBF is a prerequisite for transactivation of RNA polymerase I.

Alkaline Phosphatase↗
PubMed1991

Identification of two forms of the RNA polymerase I transcription factor UBF.

The structure of the rat homologue of the RNA polymerase I transcription factor UBF was investigated. The sequence of the protein was deduced from the sequence of overlapping cDNAs isolated from a cDNA library and from clones of the products generated by the polymerase chain reaction from random-primed, first-strand cDNA. The sequences of these clones indicated that there were two mRNAs for UBF and that the encoded proteins were similar but not identical. One form of rat UBF was essentially identical to human UBF. The second class of UBF mRNA contained an in-frame "deletion" in the coding region that results in the deletion of 37 amino acids from the predicted protein sequence. This deletion reduces the predicted molecular size of the encoded form of UBF by approximately 4400 from 89.4 kDa to 85 kDa and significantly alters the structure of one of the four HMG-1 homology regions (HMG box-2) in that form of UBF. Evidence for the existence of two mRNAs in rat cells was confirmed by a probe protection assay, and we provide evidence that other vertebrate cells contain these same two forms of UBF mRNA. These results are consistent with the observation that UBF purified from four different vertebrates migrates as two bands upon SDS/PAGE. It has been hypothesized that the HMG motifs are the DNA-binding domains of UBF. Altering one of these "boxes," as in the second form of UBF, may alter the functional characteristics of the transcription factor. Thus, the existence of different forms of UBF may have important ramifications for transcription by RNA polymerase I.

Amino Acid Sequence↗
PubMed1992

Dual role of the nucleolar transcription factor UBF: trans-activator and antirepressor.

In a reconstituted system consisting of partially purified RNA polymerase I (pol I) and the initiation factors TIF-IA, TIF-IB, and TIF-IC, the nucleolar factor UBF (upstream binding factor) stimulates transcription from the rRNA-encoding DNA (rDNA) promoter at least 50-fold. This activation is not observed at high template concentrations or in the presence of highly purified pol I. Template commitment experiments suggest that UBF activates transcription by relieving inhibition exerted by a negative-acting factor(s) in the polymerase fraction that competes for TIF-IB binding to the rDNA promoter and prevents the formation of preinitiation complexes. Using purified histone H1 bound to DNA as a model for the repressed state of the rDNA promoter, we show that UBF counteracts H1-mediated repression of pol I transcription. The implications of these findings are discussed with respect to the protein-protein and protein-DNA interactions at the rDNA promoter and the possible involvement of UBF in control of ribosomal gene transcription.

Animals↗
PubMed1991

Cloning and structural analysis of cDNA and the gene for mouse transcription factor UBF.

The gene and protein structure of the mouse UBF (mUBF), a transcription factor for mouse ribosomal RNA gene, have been determined by cDNA and genomic clones. The unique mUBF gene consists of 21 exons spanning over 13 kb. Two mRNAs coding for mUBF1 and mUBF2 having 765 a.a. and 728 a.a., respectively, are produced by an alternative splicing of exon 8. It specifies 37 amino acids constituting a part of the regions homologous to high mobility group proteins (HMG box 2). A human UBF (hUBF) cDNA obtained by polymerase chain reaction also indicates the presence of two kinds of mRNAs, the shorter form lacking the same region as mUBF2. Comparison of the cDNAs from hUBF and mUBF revealed an unusual conservation of nucleotide sequence in the 3'-terminal non-coding region. We examined the relative amounts of expression of mUBF1 and mUBF2. The eight tissues studied contained both molecular species, although mUBF2 was the predominant form of UBF. The mRNA of mUBF1 was expressed one half of the mUBF2 in quiescent mouse fibroblasts but reached the same amount in growing state.

Amino Acid Sequence↗
PubMed1992

Analysis of the rat ribosomal DNA promoter: characterization of linker-scanning mutants and of the binding of UBF.

To investigate the mechanism of transcription of the rat ribosomal DNA (rDNA) promoter, a series of 23 linker-scanning mutants were constructed and assayed in transfected CHO cells and with cell-free extracts. With minor variation, the results of the in vitro and in vivo assays paralleled one another. For example, these assays demonstrated that the mutagenesis of bases from -133 to -124, and those from -106 to -101 of the rDNA promoter significantly inhibited transcription both in vivo and in vitro. Both of these sites lie within the upstream promoter element (UPE) of the rDNA promoter. Several constructs, in particular one that mutated the bases between -49 and -45, were better promoters in vivo than the wild-type promoter. DNAse footprinting experiments with purified UBF, an RNA polymerase I transcription factor, demonstrated the importance of the bases between -106 and -101 for the binding of that factor, providing a positive correlation between the transcription experiments and the binding of UBF to the rDNA promoter.

Animals↗
PubMed1992

Characterization and immunolocalization of RNA polymerase I transcription factor UBF with anti-NOR serum in protozoa, higher plant and vertebrate cells.

We have used anti-NOR serum from a patient with rheumatoid arthritis, to study its reactivity on different phylogenetically separated species such as protozoa, higher plants, birds and mammals. The biochemical characteristics of the antigens detected after applying mono- and two-dimensional electrophoresis and electrophoretic transfers confirm that they correspond to the rRNA polymerase I transcription factor UBF. We have demonstrated the different molecular sizes, depending on the cell complexity, but the same neutral isoelectric points in whole cell extracts of the different species. We have also demonstrated an immunolocalization of this transcription factor to the fibrillar component in all the species studied. These results suggest a high conservation of UBF throughout evolution and the possibility of using this anti-NOR serum as a tool for the study of the structure, nucleolar organization and functional roles of the different nucleolar components.

Allium↗
PubMed1992

Immunostaining of nucleolus organizers in mammalian cells by a human autoantibody against the polymerase I transcription factor UBF.

A mammalian autoantigen of the nucleolus organizing regions (NORs) was identified by a human autoantibody from the serum of a rheumatoid arthritis patient. The distribution and changes of NORs during the cell cycle of mammals were followed by using this autoantiserum in indirect-immunofluorescence microscopy. In interphase cells the staining pattern indicated that the autoantigen is restricted exclusively within the nucleolus. This fluorescence appeared punctuated rather than uniform, and it was reorganized during inhibition of transcription in cells treated with actinomycin D. During mitosis, the autoantigen was detected by light microscopy at the chromosomal nucleolus organizer regions, indicating that presumably the protein remains bound to the rRNA genes. Biochemical analysis by immunoblotting showed that the NOR autoantigen consists of two polypeptides with molecular masses apparent of 90-92 kDa in all of the mammalian cell lines tested. The identity of some epitopes, recognized by this autoantibody as the ribosomal transcription factor UBF, is discussed.

3T3 Cells↗
PubMed1992

Cooperative binding of the Xenopus RNA polymerase I transcription factor xUBF to repetitive ribosomal gene enhancers.

Upstream binding factor (UBF) is a DNA-binding transcription factor implicated in ribosomal gene promoter and enhancer function in vertebrates. UBF is unusual in that it has multiple DNA-binding domains with homology to high-mobility-group (HMG) nonhistone chromosomal proteins 1 and 2. However, a recognizable DNA consensus sequence for UBF binding is lacking. In this study, we have used gel retardation and DNase I footprinting to examine Xenopus UBF (xUBF) binding to Xenopus laevis ribosomal gene enhancers. We show that UBF has a minimum requirement for about 60 bp of DNA, the size of the short enhancer variant in X. laevis. Stronger UBF binding occurs on the longer enhancer variant (81 bp) and on multiple enhancers linked head to tail. In vivo, Xenopus ribosomal gene enhancers exist in blocks of 10 alternating 60- and 81-bp repeats within the intergenic spacer. In vitro, UBF binds cooperatively to probes with 10 enhancers, with five intermediate complexes observed in titration experiments. This suggests that, on average, one UBF dimer binds every two enhancers. A single UBF dimer can produce a DNase I footprint ranging in size from approximately 30 to about 115 bp on enhancer probes of different lengths. This observation is consistent with the hypothesis that multiple DNA-binding domains or subdomains within UBF bind independently, forming more-stable interactions on longer probes.

Animals↗
PubMed1985

Effect of graded reductions in uteroplacental blood flow on the fetal lamb.

Graded reductions in uteroplacental blood flow (UBF), produced by an externally adjustable vascular occluder, were studied in unanesthetized pregnant sheep. This method was designed to simulate the physiological occurrence of reduced UBF to study its effects on fetal status. Sheep were divided into four groups in which average percent changes (+/- SE) in UBF, as measured by electromagnetic flow probes, were 7 +/- 2, -24 +/- 1, -49 +/- 1, and -63 +/- 2% for 1 h. Maternal and fetal cardiovascular (heart rate and blood pressure) and blood gas measurements of PO2 and PCO2, pH, and O2 content were determined before, during, and after recovery from UBF reduction. UBF reduction had no significant effect on maternal measurements. Changes in fetal PO2 and O2 content were directly related to the extent of vascular occlusion (P less than or equal to 0.05). Fetal pH markedly decreased after 1 h of 49% UBF reduction (P less than or equal to 0.05) and within 10 min after 63% UBF reduction (P less than or equal to 0.05). Fetal bradycardia (P less than or equal to 0.05) resulted from 63% reduction in UBF. Fetal heart rates at recovery were greater than base-line heart rate values (P less than or equal to 0.05) in the three groups whose UBF was restricted. The results of the present study suggest that the uterus of the pregnant ovine is perfused in sufficient excess to protect the fetus during short-term reductions of less than 50% UBF.

Animals↗
PubMedDate not supplied

Complementary in vivo and in vitro analyses of the interactions between the cis-acting elements of the rat rDNA promoter.

Two transcription factors, rat UBF (rUBF) and rat SL-1 are required for the efficient transcription of the rat promoter in vitro. In vitro studies have established that two broadly defined cis-acting domains, the core promoter element and the upstream promoter element, cooperate to direct correct transcription by RNA polymerase I. The ability of UBF to bind to two linker-scanning mutants of the upstream promoter element, which did not respond to the addition of UBF in in vitro transcription assays, was assessed by DNase footprinting. UBF protected the same region of the promoter in the linker-scanning mutant in BSM 129/124 as it did in the wild-type, but did not yield a typical footprint over the promoter in the linker-scanning mutant BSM 106/101. Previously we reported that promoters with mutant core promoters elements, either the guanine at -16 or -7 substituted by an adenine, were inactive in vitro unless the assays were supplemented with UBF. Those results suggested that the binding of UBF upstream of the core was required for the promotion of transcription. The interactions between the core and upstream promoter elements were assessed by constructing double mutants of the promoter. In two constructs the conserved guanines at either -16 or -7 were altered in a deletion mutant (-86) that did not respond to UBF. In a third construct the guanine at -16 in BSM 129/124 was changed to an adenine. These bidomain mutant constructs did not respond to the addition of UBF in an in vitro transcription assay, confirming that the rescue of the core promoter mutants requires an intact and functional upstream promoter element.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Deciduoma growth in the ovariectomized guinea pig: steroid hormone-mediated vascular support of endometrial differentiation.

The ability of ovarian steroid hormones to modulate experimentally induced decidual tissue (DT) growth and the associated changes in uterine blood flow rates (UBF) was examined in ovariectomized guinea pigs after uterine trauma (designated day 0 of the studies). Uteri that were exposed, but not manipulated, served as controls. Uterine and DT weights as well as UBF, rates, were subsequently recorded on either day 5 or 10 posttrauma. Oil treatment failed to induce an increase in either control or traumatized uterine weights between days 5 and 10, and trauma had no effect on UBF rates in either group. Daily progesterone (P; 2 mg) treatment induced a significant elevation in DT weight by day 10 and elevated UBF rates between days 5 and 10 relative to control values. Daily P treatment augmented by estradiol (E2; 1 microgram) therapy on days 0 and 1 induced a significant increase in DT weights and UBF rates between days 5 and 10 in both control and DT groups relative to those in oil-treated animals. Combined P and E2 (P/E2) treatment induced a moderate increase in DT weight by day 10 posttrauma and elevated UBF rates in both control and DT groups. Acute treatment (i.e. days -3 to 0) with these steroid regimens indicated that neither P nor P/E2 treatment maintained DT growth. However, day -3 to 0 treatment with P in combination with a single day 0 injection of E2 allowed for maximal DT growth by day 10 and maintained elevated UBF rates relative to control values. P/E2 treatment between days -3 and 0 also induced an increase in UBF rates in both control and DT uteri relative to those in oil-treated animals. These results indicate that E2 is essential for supporting the P-directed differentiation and proliferation of stimulated guinea pig endometrium into DT. The ability of decidualization to occur in the absence of chronic steroid support indicates that uterine sensitization for cellular differentiation in this species only requires that the endometrium be initially primed by ovarian steroid hormones, but that subsequent growth is autonomous.

Animals↗
PubMed1992

The nucleolar transcription factor mUBF is phosphorylated by casein kinase II in the C-terminal hyperacidic tail which is essential for transactivation.

UBF is a DNA binding protein which interacts with both the promoter and the enhancer of various vertebrate ribosomal RNA genes and functions as a transcription initiation factor for RNA polymerase I (pol I). We have purified murine UBF to apparent molecular homogeneity and demonstrate that its transactivating potential, but not its DNA binding activity, is modulated in response to cell growth. In vivo labelling experiments demonstrate that UBF is a phosphoprotein and that the phosphorylation state is different in growing and quiescent cells. We show that UBF is phosphorylated in vitro by a cellular protein kinase which by several criteria closely resembles casein kinase II (CKII). A major modification involves serine phosphoesterifications in the carboxy terminal hyperacidic tail of UBF. Deletions of this C-terminal domain severely decreases the UBF directed activation of transcription. The data suggest that phosphorylation of UBF by CKII may play an important role in growth dependent control of rRNA synthesis.

Amino Acid Sequence↗
PubMed1977

[Acute reduction of uterine blood flow and fetal heart rate changes in pregnant sheep near term].

Five merino sheep near term were used for acute preparations to investigate the influence of basal uterine blood flow (UBF), basal fetal oxygenation and basal fetal heart rate (FHR) on FHR changes elicited by acute reduction of UBF. The ewes were anaesthesized with pentobarbital and Alloferin was given for relaxation. Ventilation was maintained via a tracheal tube by a Starling pump. Maternal heart rate, arterial pressure and intrauterine pressure as well as fetal heart rate and umbilical artery pressure were recorded continously. Initially blood flow was recorded by cuff flow meter sequentially in both uterine arteries for a short time in order to determine total UBF and the ratio of flows. During the UBF reduction sequence flow was recorded in the artery of the gravid horn continously and measurement was corrected according to the initial ratio of flows in both arteries, presuming that this ratio would remain constant throughout the course of the experiment. Repetitive and progressive acute reduction of UBF (approximately 25%, 50% and 100% from basal UBF value) was achieved three times, lasting 120 sec each and each approximatly 12 min apart, by flow meter controlled partial or total occlusion of the aorta abdominalis of the ewe with a balloon catheter inserted into the aorta. (In some cases the lateral abdominal section was left open and the aorta was compressed manually). Before, 90 sec after the beginning and 10 min after the end of UBF reduction blood samples were taken from the fetal catheters (Fig. 3). In two preparations this sequence was repeated once after complete recovery of FHR.

Animals↗
PubMed1992

Analysis of the phosphorylation, DNA-binding and dimerization properties of the RNA polymerase I transcription factors UBF1 and UBF2.

The phosphorylation, DNA-binding and dimerization properties of both forms of the RNA polymerase I transcription factor UBF were studied and compared. Tryptic peptide maps of in vivo 32P-labeled UBF contained four phospho-peptides. Two of these peptides are predicted to derive from the serine-rich, carboxyl-terminal of UBF. This region contains nine consensus phosphorylation sites for casein kinase II, and is one of the regions phosphorylated in vitro by casein kinase II. Analysis of the DNA-binding properties of recombinant forms of UBF1 and UBF2 by Southwestern blots revealed: (1) a role for the NH2-terminal 102 amino acid domain of UBF1/UBF2 in DNA-binding; (2) the importance of the bases from -106 to -101 of the rat ribosomal DNA promoter for the binding of UBF; and (3) functional differences between UBF1 and UBF2. Glutaraldehyde cross-linking and overlay assays using recombinant forms of UBF1 and UBF2 demonstrated that the molecules can form both homodimers and heterodimers. These assays also demonstrated that the NH2-terminal 102 amino acids of UBF plays a significant role in dimerization and that other domains contribute to dimerization. The dimerization properties of recombinant forms of UBF1 and UBF2 were different, suggesting that the HMG box 2 of UBF1, which is partially deleted in UBF2, also contributes to UBF dimerization.

Animals↗
PubMed1992

Hydralazine does not restore uterine blood flow during cocaine-induced hypertension in the pregnant ewe.

Cocaine abuse is widespread, and its use by the parturient has potential significant adverse effects in both the mother and the newborn. This study was undertaken in gravid ewes to determine the effects of treatment of cocaine-induced hypertension with hydralazine (Apresoline) on the maternal and fetal cardiovascular systems, catecholamine response, blood gas and acid-base status, and uterine blood flow (UBF). Twenty-one experiments were performed in 15 chronically instrumented ewes near term gestation. After a 30-min control period, cocaine was given intravenously to all ewes for 55 min to induce and maintain increased maternal mean arterial pressure (MMAP) and reduced UBF. The sheep were randomly assigned to receive either cocaine alone (n = 11, control group) or hydralazine (n = 10, treatment group), starting 15 min after the cocaine administration. Both drugs were discontinued 55 min after the start of the cocaine administration, followed by a 35-min recovery period. In the control group, cocaine administration resulted in a 31 +/- 13% (SD) increase in MMAP (P less than 0.05) and a 26 +/- 21% reduction in UBF (P less than 0.05). In the treatment group, the initial cocaine administration resulted in a similar increase in MMAP and decrease in UBF. Hydralazine therapy restored MMAP toward baseline after 20 min of administration, but UBF remained reduced (37 +/- 17%) throughout therapy (P less than 0.05) and recovery (18 +/- 13%) (P less than 0.05). The maternal heart rate increased maximally by 121 +/- 33% (P less than 0.05) after the administration of hydralazine, compared with a 14 +/- 21% increase (P less than 0.05) in the control group.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Assembly of alternative multiprotein complexes directs rRNA promoter selectivity.

How can trans-activators with the same DNA binding specificity direct different transcriptional programs? The rRNA transcriptional apparatus offers a useful model system to address this question and to dissect the mechanisms that generate alternative transcription complexes. Here, we compare the mouse and human transcription factors that govern species-specific RNA polymerase I promoter recognition. We find that both human and mouse rRNA transcription is mediated by a specific multiprotein complex. One component of this complex is the DNA-binding transcription factor, UBF. Paradoxically, human and mouse UBF display identical DNA binding specificities even though transcription of rRNA is species specific. Promoter selectivity is conferred by a second essential factor, SL1, which, for humans, does not bind DNA independently but, instead, cooperates with UBF in the formation of high-affinity DNA-binding complexes. In contrast, mouse SL1 can selectively interact with DNA in the absence of UBF. Reconstituted transcription experiments establish that UBF and RNA polymerase I from the two species are functionally interchangeable, whereas mouse and human SL1 exhibit distinct DNA binding and transcription activities. Together, these results suggest a critical role for a specific multiprotein assembly in RNA polymerase I promoter recognition and reveal distinct mechanisms through which such complexes can generate functional diversity.

Animals↗
PubMed1990

Effects of reduced uterine blood flow on fetal cardiovascular, renal, and lung function.

Uterine blood flow (UBF) was reduced for 1 h by partially occluding the maternal aorta below the renal arteries in seven pregnant ewes (gestation age 126-134 days). Fetuses became hypoxic, acidemic, and hypercapnic. They developed hypertension (P less than 0.005) and a bradycardia (P less than 0.05). During restricted UBF, fetal hematocrit (Hct) rose (P less than 0.005) and blood volume fell in five of seven fetuses. After release of constriction, fetal Hct fell, and blood volume rose by 7.5 +/- 3.26% (P less than 0.05) relative to control. During reduced UBF, lung liquid and urine flow rates fell (P less than 0.025 and P less than 0.05, respectively). After the occluder was released, Na excretion (which did not fall significantly during reduced UBF) increased (P less than 0.05), and fractional reabsorption of Na fell (P less than 0.05). Changes in fetal blood volume (FBV) were directly related to changes in maternal lower body flow (r = 0.47, P = 0.01, n = 33), and changes in fetal Hct were inversely related to maternal flow (r = -0.635, P = 0.001). Fetal urinary Na excretion per kilogram body weight was directly related to FBV per kilogram (r = 0.44, P = 0.005, n = 40), whereas fractional reabsorption of Na was inversely related to FBV per kilogram body wt (r = 0.48, P less than 0.002, n = 39). It is concluded that reductions in UBF cause fetal hypoxemia and acidemia, which lead to changes in fetal cardiovascular function and in FBV.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1975

Umbilical blood flow and glucose uptake in lamb fetus following single umbilical artery ligation.

Umbilical blood flow (UBF) and fetal glucose uptake were measured in 5 lamb fetuses 4-20 days following single umbilical artery ligation (SUAL). The ligation was performed at 108-119 days gestation (sheep pregnancy = 145-150 days), and the blood flow and glucose uptake studies were performed as chronic preparation at 120-137 days of gestation. In comparison with 9 control subjects at matched gestation, the SUAL fetuses had significantly lower UBF (mean plus or minus SEM, 735 plus or minus 70 vs. 475 plus or minus 24 ml/min, p less than 0.01) and fetal glucose uptake (2190 plus or minus 1.6 vs. 14.8 plus or minus 1.9 mg/min, p less than 0.05). When the values were expressed per kilogram fetal weight, the UBF decreased with increasing gestational age in SUAL fetuses. In the control fetuses, the UBF increased with increasing gestational age. The fetal glucose uptake (FGU), when expressed per kilogram fetal weight, was essentially similar to that of the control subjects. The glucose gradients across the placenta were similar in both groups of fetuses. These data suggest that in lamb fetuses with placental vascular insufficiency induced by SUAL, the reduction of UBF and FGU may account for growth retardation in utero. It was also suggested that placental transfer of substrate other than glucose may also be impaired as a result o

Animals↗