Proximal sequence element factor binding and species specificity in vertebrate U6 snRNA promoters.

The Xenopus tropicalis U6 gene is very poorly transcribed both when introduced into human cells by transfection, and in human cell-free extracts. By analysis of hybrid promoters constructed from human and Xenopus sequences in various combinations, we show that species specificity is mediated by the proximal sequence elements (PSEs) of the promoters. We demonstrate the PSE-dependence of U6 transcription in a fractionated extract of HeLa cells. One of the fractions required for transcription contains an activity designated PSE-binding protein (PBP), previously shown to bind to the PSE of the mouse U6 gene. Binding of PBP to various wild-type and hybrid U6 PSE sequences correlates with their activity in transcription in HeLa cell extracts. This provides strong evidence that PBP is the PSE-binding factor involved in U6 transcription. In addition, it suggests that the differential affinities of the promoters for PBP is responsible for the observed species specificity. The divergence between U snRNA promoters in different species contrasts with the relatively strong conservation of other families of RNA polymerase II and III transcribed gene promoters. Possible mechanisms by which this diversity could be generated are discussed.

UGP--a tumor marker of gynecologic and breast malignancies? Specificity and sensitivity in pretherapeutic patients and the influence of hormonal substitution on the expression of UGP.

Urinary gonadotropin peptide (UGP) is a 10,300 Dalton peptide which is present in the urine of pregnant women, those with trophoblast disease and those with, certain nontrophoblastic malignancies. We examined the efficiency of UGP measurement at differentiating benign from malignant gynecologic and breast diseases. UGP was measured in 1355 spot urine samples from 841 patients (343 samples from 323 healthy women and women with benign gynecologic and breast diseases, 1012 samples from 518 women with gynecologic malignant diseases or breast cancer). Using a cutoff of > 3 fmol UGP/mg urinary creatinine the specificity was 97%. The sensitivity of UGP was calculated from pretherapeutically collected samples (n = 210). The sensitivity of the test for all malignancies was 26% (ovarian malignancy (n = 27) 52%, endometrial cancer (n = 25) 32%, cervical cancer (n = 49) 29%, breast cancer (n = 72) 19%, vulvar cancer and vaginal cancer (n = 12) 17% and for carcinoma in situ of the breast or the cervix (n = 20) 0%). We also found significantly higher UGP values in postmenopausal women than in premenopausal women. Hormonal substitution significantly lowered the UGP values.

TFIIA is required for in vitro transcription of mammalian U6 genes by RNA polymerase III.

Transcription factor TFIIA, defined by its role in transcription by RNA polymerase II, is also involved in RNA polymerase III transcription of mammalian U6 small nuclear RNA genes. This finding was substantiated by experimental evidence including (i) extensive copurification of an activity required for U6 transcription with TFIIA, (ii) the comparable molecular dimensions of this activity and TFIIA, (iii) the identical heat stability of both activities, and (iv) functional analyses revealing that TFIIA facilitates the interaction of TFIID with the TATA box of the U6 gene. As was shown previously for TFIID, TFIIA is the second basal transcription factor which could be demonstrated to be involved in gene expression by two different RNA polymerases.

Stimulation of lymphocytes by platelet-antibody-complexes and their role in the pathogenesis of idiopathic thrombocytopenia.

The effect of washed human platelets, platelet lysates, and platelet antibody complexes on 14C-thymidine incorporation by human lymphocytes was studied. For sensitization of platelets, HLA-specific alloantibodies as well as platelet autoantibodies were used. Lymphocytes for in vitro cultures were collected from unsensitized individuals, healthy women with proven fetomaternal immunization against HLA antigens and patients with idiopathic thrombocytopenic purpura (ITP). Unsensitized platelets have a dose-dependent inhibitory effect on the in vitro proliferation of normal lymphocytes induced by mitogens (PHA, ConA, PWM). Platelet antibody complexes (allo- and autoantibodies; allogenic and autologous lymphocyte-platelet combinations) did not stimulate 14C-thymidine incorporation. Lymphocytes from ITP patients showed a significantly reduced stimulatory response toward PHA compared to normal persons. These findings are discussed in the light of our present knowledge regarding the role of cellular immune reactions in the pathogenesis of ITP.

[Problematic blood transfusion (author's transl)]. / Die "problematische" Bluttransfusion.

Two patients were admitted with signs of haemolysis due to severe transfusion reactions. Serological investigations showed the presence of the antibodies anti-IKb and anti-Fya. After a short period of haemodialysis the patient with Kidd antibodies died. A further patient with triple antibodies (anti-c, -K, -Lub) was so pre-immunised that with an antigen frequency of over 99.9% no compatible blood could be found in Europe. By means of information from the European Central Index in Amsterdam compatible blood for the operation could be obtained from Australia. In the fourth case the antibody anti-Cob was found which had not previously been described in Germany and with which the phenotype could be determined in 1405 unselected persons in the German population. The frequency of Cob is 7.2%.

Purification of transcription factor IIIB from HeLa cells.

Transcription factor IIIB (TFIIIB), which by itself does not bind stably or specifically to DNA, was purified from cytoplasmic extracts of HeLa cells using five different chromatographic steps. This procedure yields one predominant polypeptide which represents 90% of the most highly purified preparation and shows a relative molecular mass of 60,000, when analyzed on sodium dodecyl sulfate-polyacrylamide gels. A similar value was obtained for the native protein by rate zonal centrifugation on glycerol gradients. From these data we conclude that TFIIIB from HeLa cells has a Mr of 60,000 +/- 5,000 and that it functions as a single polypeptide. Highly purified TFIIIB was required and sufficient for the specific transcription of the Xenopus laevis and human tRNA and 5 S RNA genes as well as those for VA RNA when reconstituted with RNA polymerase III and the other appropriate transcription factors.

Human transcription factor IIIC contains a polypeptide of 55 kDa specifically binding to Pol III genes.

Human transcription factor IIIC contains a 55 kDa polypeptide which specifically interacts with the Adenovirus 2 VAI gene promoter and which mimics most of the DNA binding properties of the entire factor. The specificity and affinity of this protein:DNA interaction was demonstrated by: (i) Separation of purified fractions of hTFIIIC by SDS PAGE, electrotransfer to nitrocellulose, renaturation of proteins and their subsequent binding to the VAI gene, (ii) recovery and renaturation of proteins from SDS gels and identification of a fraction of hTFIIIC with a molecular mass less than 68 kDa, which specifically binds to VAI DNA, (iii) correlating the differential binding activity of the renatured 55 kDa component of hTFIIIC to mutated Pol III promoters with the ability of the entire factor to form functional transcription complexes thereon, and finally by (iv) specific crosslinking of the 55 kDa DNA binding component of hTFIIIC to the photoaffinity labeled B-box promoter sequence of the VAI gene.

Identification of transcription factors required for the expression of mammalian U6 genes in vitro.

Transcription factors, required for the basal expression of the mouse U6 gene were identified in extracts from HeLa cells. This gene is transcribed at least four times more efficiently than its human counterpart in extracts from mouse or HeLa cells and hence provides an excellent in vitro system for the identification of transcription factors involved in the basal expression of mammalian U6 genes. At least four separate protein components were found to be required in addition to RNA polymerase III for correct synthesis of U6 RNA in vitro. These correspond to: (i) TFIIIB; (ii) a heat labile activity contained in a protein fraction enriched in TFIID; (iii) an, as yet, uncharacterized component contained in the flow-through upon rechromatography on phosphocellulose, and finally; (iv) a protein specifically binding to the mouse U6 gene promoter and transactivating its expression. Transcription factors IIIA and IIIC are not involved in mammalian U6 transcription in vitro. The U6-specific transcription factor has a molecular mass of approximately 90 +/- 10 kDa. It specifically binds to the U6 gene from bp -42 to -78 on the coding and from bp -37 to -79 on the non-coding strand thereby centrally encompassing the PSE motif of the mouse U6 promoter. The binding activity of this protein is correlated with the efficiency with which the U6 gene is transcribed in vitro, thereby indicating a crucial role of the PSE-binding protein for U6 transcription.

Human transcription factor IIIC binds to its cognate promoter sequences in a metal coordinated fashion.

Transcription factor IIIC from human cells (hTFIIIC) contains a 55 kDa polypeptide which specifically binds to the promoter of the VAI and 5S gene. This interaction can be abolished by depleting divalent metal cations from the free protein through chelation with EDTA. Prior association of the protein with its DNA-binding sequence renders the complex refractory to chelation by EDTA. Specific binding of hTFIIIC to its cognate promoter sequences--shown by electrophoretic mobility shift and DNase I protection assays--can be restored by the addition of zinc ions. In contrast to the binding of hTFIIIA to the 5S gene, which was monitored in parallel and which exclusively requires Zn2+, the binding of hTFIIIC to the VAI and 5S gene can also be reconstituted--albeit with a lower efficiency--by the transition metals Co2+, Fe2+ and Mn2+ but not by Ni2+ or Cu2+. These results show that hTFIIIC binds to its promoter sequences in a metal coordinated fashion which differs from that observed for the binding of hTFIIIA to the 5S gene.

Transcription factor IIA is inactivated during terminal differentiation of avian erythroid cells.

Avian histone H5 and alpha A-globin genes are transcribed much more efficiently in whole cell extracts derived from immature polychromatic erythrocytes than in extracts from mature duck erythrocytes. We found that these differential activities are detectable only if assayed with promoters containing a functional TATA box. The addition of either highly purified human or recombinant yeast transcription factor IIA (TFIIA) to extracts from mature erythrocytes resulted in a significant increase in transcription from TATA-containing promoters, whereas transcription from TATA-less promoters remained unaffected. Moreover, the activity of TFIIA was found to be reduced in extracts from mature erythrocytes. These data support the proposition that inactivation of TFIIA may contribute to a general repression of gene activity in avian erythrocytes, and only those genes with alternative mechanisms of initiation complex formation continue to be expressed in these cells. In the case of the histone H5 gene, such an alternative mechanism could be mediated via the interaction between duck erythrocyte upstream stimulating factor and TFIID.

Mammalian transcription factor PBP. Characterization of its binding properties to the proximal sequence element of U6 genes.

The DNA binding properties of human transcription factor PBP, which specifically binds to the proximal sequence element of mammalian U6 genes and which plays a pivotal role during their transcription, were analyzed both qualitatively and quantitatively. As a prerequisite, we analyzed the optimal conditions for DNA binding of the PBP by assaying the stability of the interaction against increasing concentrations of salt, dithiothreitol, and heparin. The protein, which does not induce DNA bending, has a characteristic sensitivity against elevated temperatures and precipitously loses activity between 41 and 43 degrees C, a property which can be used for selective inactivation of the protein. Subjection of the PBP to limited proteinase K treatment showed that the protein consists of at least two functional domains, one of which is required for DNA binding. The PBP binds to the PSE with a much higher specific equilibrium constant (Ks = 1.33 x 10(11) M-1) than to nonspecific DNA (Kn = 1.18 x 10(5) M-1). The association and dissociation rates of PBP.PSE interactions were quantitatively determined by kinetic analyses. The pronounced lag phase during the initiation reaction of mammalian U6 transcription in vitro is probably correlated with the slow binding of the PBP to its target sequence. Once formed, however, the PBP.PSE complex is very stable and has a much lower dissociation (kd = 1.84 x 10(-5) s-1) than association rate constant (ka = 0.18 x 10(6) M-1 s-1). Collectively, the results demonstrate that the PSE binding protein stably associates with a high affinity to its cognate promoter sequence, and this process represents one of the primary events in the formation of the preinitiation complex on the U6 gene. Finally, we analyzed the effect of individual base pair mutations within mammalian U6 PSE sequences on the binding of the PBP.

Transcription factor IIA stimulates the expression of classical polIII-genes.

Protein fractions containing TFIIA, a transcription factor known to be involved in transcription initiation by RNA polymerase II and 5'-regulated polymerase III genes (e.g. U6), were tested for their role in in vitro transcription of classical pol III genes. These fractions were shown to stimulate a basal transcription system, reconstituted from highly purified fractions hTFIIIB and hTFIIIC. We demonstrate that this stimulating activity isolated from HeLa cells coelutes over at least six chromatographic steps with hTFIIA. Moreover the native molecular mass and the stability of this activity against heat treatment are comparable to those of hTFIIA. Finally we show that recombinant TFIIA from Saccharomyces cerevisiae can substitute for the human factor in pol III transcription in vitro which proves that TFIIA is also involved in the efficient expression of classical pol III genes.

[Discontinuous cell separation as depletion therapy in chronic leukemias]. / Die diskontinuierliche Zellseparation als depletorische Therapie bei chronischen Leukämien.

Blood cell separators have been successfully used in the treatment of chronic leukaemias. With the intermittent flow system Haemonetics model 30, we found it important to collect 150-300 ml buffy coat/cycle in order to obtain a substantial depletory effect. In 2 patients with chronic myeloid leukaemia on short-term leukapheresis and in 1 patient with prolymphocytic leukaemia on long-term leukapheresis we found a definite decrease in the WBC count. 2 of them experienced a transitory improvement of their disease. Only minor side-effects occurred. However, the loss of erythrocytes and plasma albumin necessitated replacement therapy. From our experience the intermittent flow blood cell separator can be successfully used in the treatment of well-defined forms of chronic leukaemia.

Physical and immunological characterization of human transcription factor IIIA.

Human transcription factor IIIA (htFIIIA), specifically required for transcription of the gene for 5S ribosomal RNA has been characterized with respect to some of its physical, immunological and functional properties. TFIIIA from HeLa cells, which selectively binds 5S RNA, is a monomer of approximately 35 kDa with a Stokes' radius of approximately 2.65 nm and a sedimentation coefficient of approximately 2.8 S. These values indicate that the human protein is of rather globular shape and hence diverges not only in molecular mass but also in most of the molecular properties from its highly asymmetric counterpart in Xenopus laevis oocytes. By raising specific polyclonal antibodies against hTFIIIA it was shown in Western immunoblots that there was no cross-reaction between anti-hTFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA antibodies and the amphibian protein. Conversely, monoclonal antibodies against three domains of X. laevis TFIIIA did not cross-react with the human transcription factor. The polyclonal antisera raised against hTFIIIA specifically neutralized binding of the human transcription factor to 5S DNA and abolished in vitro transcription of 5S RNA but these antibodies were unable to inhibit 5S RNA synthesis in cellular extracts from Xenopus, Drosophila or yeast cells. Finally, the species variation of TFIIIA could be substantiated by electrophoretic mobility shift assays revealing preferential binding of hTFIIIA to the homologous 5S RNA gene.

Purification of human transcription factor IIIC and its binding to the gene for ribosomal 5S RNA.

Transcription factor hTFIIIC was purified from cytoplasmic extracts of HeLa cells using four different chromatographic steps. This procedure yields a protein fraction which actively supports transcription in reconstitution assays and contains five major polypeptide chains with a molecular mass ranging from 25 to 250 kDa as estimated by SDS-PAGE and silver staining. In this fraction a polypeptide with a molecular mass of approximately 110 kDa could be identified as a specific DNA-binding component of hTFIIIC. By electrophoretic mobility shift and footprinting analyses it could be demonstrated that purified hTFIIIC binds specifically to the 5S gene. The protected region encompasses the A-Box promoter element and flanking sequences extending toward the 5'-proximal end of the gene. By addition of hTFIIIC to preformed TFIIIA/5S DNA complexes, we observe an additive effect of both factors on the footprint boundaries.

Purification of human transcription factor IIIA and its interaction with a chemically synthesized gene encoding human 5 S rRNA.

Transcription factor IIIA (TFIIIA) was purified from cytoplasmic extracts of HeLa cells by developing a simple and efficient procedure employing phosphocellulose under widely differing ionic conditions followed by affinity chromatography on immobilized human 5 S genes. This procedure yielded a fraction containing human TFIIIA activity and a protein of 35 kDa as its major component. Moreover, we succeeded in renaturing the activity of human transcription factor IIIA (hTFIIIA) isolated after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in identifying a polypeptide of 35 kDa with the transcription activity. This value differs from that reported for Xenopus TFIIIA. It could be demonstrated by footprinting analyses that hTFIIIA specifically binds to the internal control region of the human 5 S rRNA gene. The limits of protection slightly differ at the 3' border of the internal control region from those imprinted by Xenopus TFIIIA on the same gene. Comparative footprint analyses of hTFIIIA on the human and frog somatic 5 S rRNA gene, measured in titration, competition, and salt-stability experiments, demonstrated a higher affinity of the human factor to the homologous gene. These results, together with the difference in molecular mass of these functionally analogous proteins, reemphasize the importance of homologous systems for the analysis of mechanisms involved in gene regulation.