Phosphorylation of UBF at serine 388 is required for interaction with RNA polymerase I and activation of rDNA transcription.

Modulation of the activity of the upstream binding factor (UBF) plays a key role in cell cycle-dependent regulation of rRNA synthesis. Activation of rDNA transcription on serum stimulation requires phosphorylation of UBF at serine 484 by G(1)-specific cyclin-dependent kinase (cdk)/cyclin complexes. After G(1) progression UBF is phosphorylated at serine 388 by cdk2/cyclin E and cdk2/cyclin A. Conversion of serine 388 to glycine abolishes UBF activity, whereas substitution by aspartate enhances the transactivating function of UBF. Protein-protein interaction studies reveal that phosphorylation at serine 388 is required for the interaction between RNA polymerase I and UBF. The results suggest that phosphorylation of UBF represents a powerful means of modulating the assembly of the transcription initiation complex in a proliferation- and cell cycle-dependent fashion.

Overlapping functions of the pRb family in the regulation of rRNA synthesis.

The "pocket" proteins pRb, p107, and p130 are a family of negative growth regulators. Previous studies have demonstrated that overexpression of pRb can repress transcription by RNA polymerase (Pol) I. To assess whether pRb performs this role under physiological conditions, we have examined pre-rRNA levels in cells from mice lacking either pRb alone or combinations of the three pocket proteins. Pol I transcription was unaffected in pRb-knockout fibroblasts, but specific disruption of the entire pRb family deregulated rRNA synthesis. Further analysis showed that p130 shares with pRb the ability to repress Pol I transcription, whereas p107 is ineffective in this system. Production of rRNA is abnormally elevated in Rb(-/-) p130(-/-) fibroblasts. Furthermore, overexpression of p130 can inhibit an rRNA promoter both in vitro and in vivo. This reflects an ability of p130 to bind and inactivate the upstream binding factor, UBF. The data imply that rRNA synthesis in living cells is subject to redundant control by endogenous pRb and p130.

Acetylation of TAF(I)68, a subunit of TIF-IB/SL1, activates RNA polymerase I transcription.

Mammalian rRNA genes are preceded by a terminator element that is recognized by the transcription termination factor TTF-I. In exploring the functional significance of the promoter-proximal terminator, we found that TTF-I associates with the p300/CBP-associated factor PCAF, suggesting that TTF-I may target histone acetyltransferase to the rDNA promoter. We demonstrate that PCAF acetylates TAF(I)68, the second largest subunit of the TATA box-binding protein (TBP)-containing factor TIF-IB/SL1, and acetylation enhances binding of TAF(I)68 to the rDNA promoter. Moreover, PCAF stimulates RNA polymerase I (Pol I) transcription in a reconstituted in vitro system. Consistent with acetylation of TIF-IB/SL1 being required for rDNA transcription, the NAD(+)-dependent histone deacetylase mSir2a deacetylates TAF(I)68 and represses Pol I transcription. The results demonstrate that acetylation of the basal Pol I transcription machinery has functional consequences and suggest that reversible acetylation of TIF-IB/SL1 may be an effective means to regulate rDNA transcription in response to external signals.

Complete sequence of the 24-mer hemocyanin of the tarantula Eurypelma californicum. Structure and intramolecular evolution of the subunits.

Hemocyanins are large oligomeric respiratory proteins found in many arthropods and molluscs. The hemocyanin of the tarantula Eurypelma californicum is a 24-mer protein complex with molecular mass of 1, 726,459 Da that consists of seven different polypeptides (a-g), each occupying a distinct position within the native molecule. Here we report the complete molecular structure of the E. californicum hemocyanin as deduced from the corresponding cDNAs. This represents the first complex arthropod hemocyanin to be completely sequenced. The different subunits display 52-66% amino acid sequence identity. Within the subunits, the central domain, which bears the active center with the copper-binding sites A and B, displays the highest degree of identity. Using a homology modeling approach, the putative three-dimensional structure of individual subunits was deduced and compared. Phylogenetic analyses suggest that differentiation of the individual subunits occurred 400-550 million years ago. The hemocyanin of the stemline Chelicerata was probably a hexamer built up of six distinct subunit types a, b/c, d, e, f, and g, whereas that of the early Arachnida was originally a 24-mer that emerged after the differentiation of subunits b and c.

Phosphorylation by G1-specific cdk-cyclin complexes activates the nucleolar transcription factor UBF.

Transcription of rRNA genes by RNA polymerase I increases following serum stimulation of quiescent NIH 3T3 fibroblasts. To elucidate the mechanism underlying transcriptional activation during progression through the G1 phase of the cell cycle, we have analyzed the activity and phosphorylation pattern of the nucleolar transcription factor upstream binding factor (UBF). Using a combination of tryptic phosphopeptide mapping and site-directed mutagenesis, we have identified Ser484 as a direct target for cyclin-dependent kinase 4 (cdk4)-cyclin D1- and cdk2-cyclin E-directed phosphorylation. Mutation of Ser484 impairs rDNA transcription in vivo and in vitro. The data demonstrate that UBF is regulated in a cell cycle-dependent manner and suggest a link between G1 cdks-cyclins, UBF phosphorylation and rDNA transcription activation.

Mitotic silencing of human rRNA synthesis: inactivation of the promoter selectivity factor SL1 by cdc2/cyclin B-mediated phosphorylation.

We have used a reconstituted cell-free transcription system to investigate the molecular basis of mitotic repression of RNA polymerase I (pol I) transcription. We demonstrate that SL1, the TBP-containing promoter-binding factor, is inactivated by cdc2/cyclin B-directed phosphorylation, and reactivated by dephosphorylation. Transcriptional inactivation in vitro is accompanied by phosphorylation of two subunits, e.g. TBP and hTAFI110. To distinguish whether transcriptional repression is due to phosphorylation of TBP, hTAFI110 or both, SL1 was purified from two HeLa cell lines that express either full-length or the core domain of TBP only. Both TBP-TAFI complexes exhibit similar activity and both are repressed at mitosis, indicating that the variable N-terminal domain which contains multiple target sites for cdc2/cyclin B phosphorylation is dispensable for mitotic repression. Protein-protein interaction studies reveal that mitotic phosphorylation impairs the interaction of SL1 with UBF. The results suggest that phosphorylation of SL1 is used as a molecular switch to prevent pre-initiation complex formation and to shut down rDNA transcription at mitosis.

Equine infectious anemia virus transactivator is a homeodomain-type protein.

Lentiviral transactivator (Tat) proteins are essential for viral replication. Tat proteins of human immunodeficiency virus type 1 and bovine immunodeficiency virus form complexes with their respective RNA targets (Tat responsive element, TAR), and specific binding of the equine anemia virus (EIAV) Tat protein to a target TAR RNA is suggested by mutational analysis of the TAR RNA. Structural data on equine infectious anemia virus Tat protein reveal a helix-loop-helix-turn-helix limit structure very similar to homeobox domains that are known to bind specifically to DNA. Here we report results of gel-shift and footprinting analysis as well as fluorescence and nuclear magnetic resonance spectroscopy experiments that clearly show that EIAV Tat protein binds to DNA specifically at the long terminal repeat Pu.1 (GTTCCTGTTTT) and AP-1 (TGACGCG) sites, and thus suggest a common mechanism for the action of some of the known lentiviral Tat proteins via the AP-1 initiator site. Complex formation with DNA induces specific shifts of the proton NMR resonances originating from amino acids in the core and basic domains of the protein.

Mechanism of repression of RNA polymerase I transcription by the retinoblastoma protein.

The retinoblastoma susceptibility gene product pRb restricts cellular proliferation by affecting gene expression by all three classes of nuclear RNA polymerases. To elucidate the molecular mechanisms underlying pRb-mediated repression of ribosomal DNA (rDNA) transcription by RNA polymerase I, we have analyzed the effect of pRb in a reconstituted transcription system. We demonstrate that pRb, but not the related protein p107, acts as a transcriptional repressor by interfering with the assembly of transcription initiation complexes. The HMG box-containing transcription factor UBF is the main target for pRb-induced transcriptional repression. UBF and pRb form in vitro complexes involving the C-terminal part of pRb and HMG boxes 1 and 2 of UBF. We show that the interactions between UBF and TIF-IB and between UBF and RNA polymerase I, respectively, are not perturbed by pRb. However, the DNA binding activity of UBF to both synthetic cruciform DNA and the rDNA promoter is severely impaired in the presence of pRb. These studies reveal another mechanism by which pRb suppresses cell proliferation, namely, by direct inhibition of cellular rRNA synthesis.

The superficial femoral vein as arterial substitute in infections of the aortoiliac region.

In situ autogenous reconstruction is an alternative therapy for abdominal aortic prosthetic graft infection. We have used the superficial femoral vein (SFV) as an arterial substitute for proximal aortic anastomosis in seven patients. Six patients presented with aortic perigraft infections and one had a mycotic aneurysm of the infrarenal aorta with a primary aortoenteric fistula. There were no intraoperative but two postoperative deaths. During follow-up (mean: 19.8 months), one patient died at 5 months unrelated to his preceding vascular procedures; the others were doing well with patient SFV grafts 6-36 months after autogenous aortoiliac reconstruction. The SFV is a valuable donor vessel for autogenous reconstruction in the management of aortoiliac prosthetic graft infection. We explain both perioperative deaths in our treatment group with respect to the extent of the underlying infection and the virulence of the causative organism.

High mobility group 1 protein is not stably associated with the chromosomes of somatic cells.

High mobility group 1 (HMG1) protein is an abundant and conserved component of vertebrate nuclei and has been proposed to play a structural role in chromatin organization, possibly similar to that of histone H1. However, a high abundance of HMG1 had also been reported in the cytoplasm and on the surface of mammalian cells. We conclusively show that HMG1 is a nuclear protein, since several different anti-HMG1 antibodies stain the nucleoplasm of cultured cells, and epitope-tagged HMG1 is localized in the nucleus only. The protein is excluded from nucleoli and is not associated to specific nuclear structures but rather appears to be uniformly distributed. HMG1 can bind in vitro to reconstituted core nucleosomes but is not stably associated to chromatin in live cells. At metaphase, HMG1 is detached from condensed chromosomes, contrary to histone H1. During interphase, HMG1 readily diffuses out of nuclei after permeabilization of the nuclear membranes with detergents, whereas histone H1 remains associated to chromatin. These properties exclude a shared function for HMG1 and H1 in differentiated cells, in spite of their similar biochemical properties. HMG1 may be stably associated only to a very minor population of nucleosomes or may interact transiently with nucleosomes during dynamic processes of chromatin remodeling.

[The superficial femoral vein in aorto-iliac position--suitable as autogenous vascular replacement in deep prosthesis infection?]. / Vena femoralis superficialis in aorto-iliacaler Position--als autogener Gefässersatz beim tiefen Protheseninfekt geeignet?

The current standard therapy of aortic graft infection is connected with a high incidence of operative and late complications. The usefulness of autogenous tissue revascularization techniques as promising therapeutic alternatives is limited by the complexity of these procedures and by the lack of suitable donor vessels. Since several authors have shown that serious venous stasis of the donor leg does not occur after superficial femoral vein (SFV) resection, we have started to use this deep leg vein for arterial in situ reconstructions in the treatment of aortic graft infection. During the past 3 years seven patients have received autogenous SFV grafts in this way. There were no intraoperative deaths. Two critically ill patients with severe Pseudomonas sepsis and extensive retroperitoneal abscess each died on the 4th postoperative day, due to acute massive bleeding in the operating area. In all of the five survivors infection could be eradicated; only one limb had to be amputated. Mild to moderate transient swelling of the lower extremity without stasis was regularly seen after SFV resection. During follow-up one patient died of cardiac arrest 5 months postoperatively. The remaining four patients are still alive with patent SFV grafts at 6, 22, 30 and 36 months after the operation.

Activation of mammalian ribosomal gene transcription requires phosphorylation of the nucleolar transcription factor UBF.

The nucleolar factor UBF is phosphorylated by casein kinase II (CKII) at serine residues within the C-terminal acidic domain which is required for transcription activation. To investigate the biological significance of UBF modification, we have compared the trans-activating properties of cellular UBF and recombinant UBF expressed in Escherichia coli. Using a variety of assays we demonstrate that unphosphorylated UBF is transcriptionally inactive and has to be phosphorylated at multiple sites to stimulate transcription. Examination of cDNA mutants in which the serine residues within the C-terminal domain were altered by site-directed mutagenesis demonstrates that CKII-mediated phosphorylations of UBF contribute to, but are not sufficient for, transcriptional activation. Besides CKII, other cellular protein kinases phosphorylate UBF at distinct sites in a growth-dependent manner. The marked differences in the tryptic peptide maps of UBF from growing and serum-starved cells suggest that alterations in the degree of UBF phosphorylation may modulate rRNA synthetic activity in response to extracellular signals.

[Aorto-intestinal fistula as a possible cause of endoscopically undetermined gastrointestinal hemorrhage]. / Aortointestinale Fistel als mögliche Ursache einer endoskopisch unklaren gastrointestinalen Blutung.

Primary aorto-enteric fistulae are rare, mostly described as atypical first manifestation of an abdominal aortic aneurysm. Spread of elective aortic surgery led to increased appearance of secondary aorto-enteric fistulae as a typical postoperative complication. Gastrointestinal bleeding with endoscopical unclear findings in a patient with aortic aneurysm or history of aortic repair points towards an aorto-enteric fistula. While preoperative diagnosis is not possible in most instances, the proof of an anastomotic aneurysm and/or aortic graft infection hardens the suspected diagnosis of an aorto-enteric fistula decisively. The finding of coincidental mucosal lesions at gastroscopy may not mislead to give up the exclusion of an aorto-enteric fistula, possibly by explorative laparotomy, if suspicion is well-founded. In the present article nine cases of aorto-enteric fistulae treated at the Surgical University Clinic Würzburg between 1982 and 1993 are analyzed retrospectively. Topical questions of diagnosis and therapy are discussed.

[Open thrombendarterectomy of the carotid arteries with routine intraluminal shunt implantation, a reliable method for decreasing the risk of apoplexy--results of 11 years experience with 546 consecutive elective interventions]. / Die offene Thrombendarteriektomie der Carotiden mit routinemässiger intraluminärer Shunteinlage, eine sichere Methode zur Verminderung des Apoplexrisikos--Ergebnisse aus 11 Jahren Erfahrung mit 546 aufeinanderfolgenden Elektiveingriffen.

The operative removal of haemodynamical significant carotid artery stenosis by endarterectomy nowadays is one of the vascular surgical standard procedures. Purpose of the operation is prevention of ischemic strokes. For a long-term prognostic advantage the patient has to take the risk of perioperative mortality and morbidity. While efforts are being made to minimize this risk, the question of optimal surgical strategy has not yet finally been solved. Since 1982 in our hospital all carotid endarterectomies are carried out with routine insertion of an intraluminal shunt. The distal intima step of the internal carotid artery is secured by a running suture and closure of the longitudinal arteriotomy is accomplished by dacron patch plasty. In this manner 546 successive operations have been performed under general anaesthesia until 1993. Intra- and postoperative mortality was 0.9% with an ischemic cerebral infarction rate of 1.8%. According to the preoperative stage of cerebrovascular insufficiency the frequencies for mortality and perioperative ischemic stroke were 0.6% and 1.3% for CVI I, 0.4% and 0.7% for CVI II and 2.8% and 5.7% for CVI IV. Apart from perioperative mortality for patients with CVI IV, these complication rates are clearly below the suggested limits of the Ad hoc Committee on Carotid Surgery Standards by the Stroke Council of the American Heart Association. Routine use of a temporary, intraluminal shunt in carotid artery operations therefore can be considered as a safe measure, with complication rates still not underbid by those achieved with intraoperative cerebral monitoring and selective shunting.

Functional differences between the two splice variants of the nucleolar transcription factor UBF: the second HMG box determines specificity of DNA binding and transcriptional activity.

The nucleolar transcription factor UBF consists of two proteins, UBF1 and UBF2, which originate by alternative splicing. Here we show that deletion of 37 amino acids within the second of five HMG box motifs in UBF2 is important for the dual role of UBF as transcriptional activator and antirepressor. UBF1 is a potent antirepressor and transcriptional activator, whereas the ability of UBF2 to counteract histone H1-mediated repression and to stimulate ribosomal gene transcription both in vivo and in vitro is at least one order of magnitude lower. The difference in transcriptional activity between UBF1 and UBF2 is due to their different binding to the ribosomal gene promoter and enhancer. Apparently, the presence of an intact HMG box2 modulates the sequence-specific binding of UBF to rDNA control elements. However, the interaction of UBF with rDNA does not entirely depend on sequence recognition. Both UBF isoforms bind efficiently to four-way junction DNA, indicating that they recognize defined DNA structures rather than specific sequences. The results demonstrate that the HMG boxes are functionally diverse and that HMG box2 plays an important role in specific binding of UBF to rDNA.

The RNA polymerase I-specific transcription initiation factor UBF is associated with transcriptionally active and inactive ribosomal genes.

We have characterized an anti-NOR (nucleolar organizer region) serum (P419) from a patient with rheumatoid arthritis and show that it contains antibodies directed against the RNA polymerase I-specific transcription initiation factor UBF. This serum reacts with UBF from a variety of vertebrate cells as revealed both by immunoblotting and by indirect immunofluorescence. We have used the P419 serum to study the intracellular localization of this transcription factor at the light and electron microscopic level. In interphase cells, UBF exhibits a pronounced punctate pattern and is found to be associated with necklace-like structures, which appear to reflect the transcriptionally active state of the nucleolus. Inhibition of rRNA synthetic activity caused either by nutritional starvation or by actinomycin D treatment resulted in a marked decrease in the number and in a significant increase in the size of UBF-positive granules. Under all experimental conditions applied, UBF was exclusively found within the nucleolus and was not released into the nucleoplasm or cytoplasm. During mitosis, UBF was found to be concentrated at the chromosomal NOR indicating that a significant quantity, if not all, of this factor remains bound to the ribosomal transcription units. From this we conclude that UBF is associated both with transcriptionally active and inactive rRNA genes and, therefore, changes in the intracellular localization of UBF are very likely not involved in rDNA transcription regulation.

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.

Arthropod hemocyanins. Molecular cloning and sequencing of cDNAs encoding the tarantula hemocyanin subunits a and e.

cDNA clones comprising the entire coding region of two out of the seven heterogeneous subunits of hemocyanin from the tarantula, Eurypelma californicum, were isolated from four cDNA libraries constructed from total RNA from the heart tissue of single spiders. Hybridization was first carried out using a tarantula hemocyanin subunit e partial cDNA, and several positive clones were isolated, including one containing a 2.2-kilobase full-length cDNA (lambda M1). The cDNA comprises an open reading frame for 623 amino acids, 34 nucleotides of the 5'noncoding region, and 286 nucleotides of the 3'-noncoding region. To select for other hemocyanin subunits, two 17-mer oligonucleotide mixtures, corresponding to the conserved regions in the copper A and copper B oxygen-binding site of chelicerate hemocyanins, were used as probes. Among the positive clones obtained, full-length cDNAs coding for subunit a were identified. The cDNA sequence determined from clone lambda K1 provides an open reading frame coding for 630 amino acids and includes the 5'- and 3'-noncoding regions. Northern blot analysis revealed single transcripts for subunits a and e, each 2.3 kilobases long. The cDNAs for subunits a and e were both found to lack any leader peptide sequence. This supports the idea that the mature protein accumulates in the cytoplasm and is released by cell rupture.

Characterization of the gene encoding the hemocyanin subunit e from the tarantula Eurypelma californicum.

The gene for the hemocyanin subunit e of the tarantula Eurypelma californicum has been isolated from a genomic phage library by using a corresponding cDNA clone as a probe. The transcriptional unit spans a chromosomal region of about 55 kilobase pairs (kbp). The gene consists of nine exons that are separated by large introns. The intron/exon boundaries were determined by direct comparison of genomic and cDNA sequences. A putative promoter region ("TATA" box, reversed "CAAT" box) 100 bp 5' to the translational initiation codon strongly suggests the presence of a functional gene. The 3' flanking region carries the polyadenylylation signal (AATAAA) and several conserved structures for the 3' splicing of the pre-mRNA. A comparison of the gene architecture of the subunit e gene with the three-dimensional structure of the arthropod hemocyanin subunit shows a good correspondence with the division of the subunit into three domains (two exons coding for the first, three coding for the second, and four coding for the third domain). The relationship to molluscan hemocyanins, different tyrosinases, and the larval serum proteins is discussed.

cDNA cloning and sequencing of tarantula hemocyanin subunits.

Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.