Interaction of the yeast Pso5/Rad16 and Sgs1 proteins: influences on DNA repair and aging.

The interaction trap method was used to isolate putative binding partners of Rad16/Pso5, a protein responsible for repair of silent DNA. One of the interactors found was Sgs1, a DNA helicase influencing the life span of Saccharomyces cerevisiae, with homology to the human BLM, WRN and RECQL4 proteins. Using the same fusion proteins from the two-hybrid screening, we show evidence that both proteins also interact in vitro. We tested isogenic strains, containing mutant alleles of the two genes in single and double mutant combination, for phenotypic similarity. Life span in sgs1Delta single and sgs1Delta rad16Delta double mutants is about 40% of that of WT, and the rad16/pso5Delta single mutant also had its life span reduced to 75%. Sensitivity to different mutagens, whose lesions are poorly repaired in rad16/pso5Delta mutants, was tested in sgs1Delta mutants. The sgs1Delta conferred sensitivity to MMS, H2O2 and was moderately sensitive to UV(254nm) (UVC) and 4-NQO. An epistatic interaction between rad16 and sgs1 mutations after UVC, 4-NQO and H2O2 was observed. Moreover, we found that in a top3 background, functional Sgs1p and Rad16p apparently channel MMS, 4-NQO and H2O2 induced lesions into aberrant DNA repair. Our results demonstrate that Sgs1 is not only involved in genome stability, somatic recombination and aging, but is also implicated, together with Rad16/Pso5, in the repair of specific DNA damage.

Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.

MAGE-11 protein is highly conserved in higher organisms and located predominantly in the nucleus.

The MAGE-11 gene belongs to a family whose products were identified first in tumor tissue. The MAGE-11 gene product has not been characterized in detail. We have isolated MAGE-11 cDNA from HeLa cells and confirmed the presence of MAGE-11 protein and of at least 2 other MAGE proteins (MAGE-1 and MAGE-3) in this cell line. Monoclonal antibodies (MAbs), obtained by using a GST-MAGE-11 fusion protein, detect MAGE-11 protein in HeLa cells as a 48 kDa protein. In contrast to other known proteins of the MAGE family, MAGE-11 is found mainly in the nucleus. Immunoprecipitation out of whole-cell extracts from different species reveals that MAGE-11 protein is highly conserved among mammalian cells, suggesting a conserved and important function.

[Human cloning. The biological fundamentals and an ethical-legal assessment]. / La clonación humana. Fundamentos biológicos y valoración ético-jurídica.

Following a description of the cloning process and how this might be used in humans, the authors examine the possibility of human cloning in the light of recognised ethical principles. They also address the question of whether current national and international laws are sufficient to prevent such practices.

RNA aptamers specifically interact with the prion protein PrP.

We have isolated RNA aptamers which are directed against the recombinant Syrian golden hamster prion protein rPrP23-231 (rPrPc) fused to glutathione S-transferase (GST). The aptamers did not recognize the fusion partner GST or the fusion protein GST::rPrP90-231 (rPrP27-30), which lacks 67 amino acids from the PrP N terminus. The aptamer-interacting region of PrPc was mapped to the N-terminal amino acids 23 to 52. Sequence analyses suggest that the RNA aptamers may fold into G-quartet-containing structural elements. Replacement of the G residues in the G quartet scaffold with uridine residues destroyed binding to PrP completely, strongly suggesting that the G quartet motif is essential for PrP recognition. Individual RNA aptamers interact specifically with prion protein in brain homogenates from wild-type mice (C57BL/6), hamsters (Syrian golden), and cattle as shown by supershifts obtained in the presence of anti-PrP antibodies. No interaction was observed with brain homogenates from PrP knockout mice (prn-p(0/0)). Specificity of the aptamer-PrP interaction was further confirmed by binding assays with antisense aptamer RNA or a mutant aptamer in which the guanosine residues in the G tetrad scaffold were replaced by uridine residues. The aptamers did not recognize PrP27-30 in brain homogenates from scrapie-infected mice. RNA aptamers may provide a first milestone in the development of a diagnostic assay for the detection of transmissible spongiform encephalopathies.

Rapid amplification of cDNA ends (RACE) improves the PCR-based isolation of immunoglobulin variable region genes from murine and human lymphoma cells and cell lines.

The isolation of rearranged immunoglobulin (Ig) variable region (V) genes is usually performed by PCR with consensus primers binding to conserved regions within the V sequences. However, the isolation of Ig genes by this method is hampered in 15-35% by technical difficulties, mostly mismatches of oligonucleotide primers to V sequences. In order to obtain DNA sequences from V heavy chain (VH) genes which could not be amplified with consensus primers, we used a modified PCR technique, the rapid amplification of cDNA ends (RACE) PCR in combination with new heavy chain constant region primers for the isolation of human and murine VH genes. In comparison, consensus primer PCR with different sets of previously published oligonucleotide primers was used. Both methods were applied to isolate VH genes from murine B cell lymphoma (A20 and BCL1), myeloma (NS1) and hybridoma (SP6) cell lines and from freshly isolated human chronic lymphocytic leukemia and lymphoma cells. RACE PCR allowed the amplification and subsequent cloning of the complete VH gene in all cases. In contrast, consensus primer PCR failed to isolate the VH sequence of the murine A20 cell line; this was explained by a mismatch of consensus primers with VH sequences. When both PCR methods amplified VH sequences, the DNA sequences obtained were identical. Taken together, RACE PCR represents a reliable and versatile method for the isolation of VH genes from human and murine lymphoma cells, in particular if consensus primer PCR fails.

Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response.

Gene transfer of the costimulatory molecules B7-1 and B7-2 induces a potent antitumor immune response in a variety of tumor models. B cell neoplasms including multiple myeloma (MM) often show little or no expression of B7 antigens; they are therefore a potential target for this approach. To increase the expression of human B7 genes in MM cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (rAAV). The resulting recombinant viruses rAAV/B7-1, rAAV/B7-2 and rAAV/Neo were used to transduce the MM cell lines LP-1 and RPMI 8226. This allowed the transduction of up to 80% of LP-1 cells 4 days after infection with purified rAAV particles. The response of human allogeneic T cells to rAAV/B7-1 and rAAV/B7-2 transduced, gamma-irradiated LP-1 cells was assessed by [3H]thymidine incorporation, by RT-PCR-based detection of immunostimulatory cytokine transcripts and by ELISA quantification of cytokines in the supernatant. Stimulation of T cells with rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells resulted in an up to 10-fold increase of T cell proliferation when compared with LP-1 cells transduced with rAAV/Neo. Similar results were obtained with RPMI 8226 cells. Both rAAV/B7-1 and rAAV/B7-2 transduced LP-1 cells stimulated the T cell secretion of IL-2 and IFN-gamma. Furthermore, [51Cr] release assays showed that rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells induced a cytolytic T cell (CTL) response, in contrast to LP-1 cells transduced with rAAV/Neo. In all assays, the effects of rAAV/B7-1 and rAAV/B7-2 were similar. Taken together, the results show that rAAV-mediated transfer of B7 genes into MM cell lines is able to enhance the antitumor T cell response and to elicit a cytolytic T cell response.

Specific repression of Tax trans-activation by TAR RNA-binding protein TRBP.

The human T-cell leukemia virus type 1 (HTLV-1)-encoded Tax protein activates transcription from the long terminal repetition via association with host cellular factors. In this study, we searched for cellular proteins that interact with Tax and modulate its activity by using the yeast two-hybrid system. One of the strongest interactors was found to be identical with TRBP, which was previously shown to bind to the RNA encoded by the Tat response element of human immunodeficiency virus type 1. Interactions are demonstrated with Escherichia coli-expressed proteins in vitro and in mammalian cells, using one- and two-hybrid systems, and with antibodies that coprecipitate Tax and TRBP at physiological TRBP concentrations. Moreover, TRBP, when directed into the cytoplasm, is capable of preventing transport of Tax into the nucleus. A 60-amino-acid polypeptide suffices for binding to Tax. TRBP inhibits activation of transcription by both Tax and GAL4-Tax fusion proteins. Inhibition is specific for Tax and is not seen with the other activators tested. Our data are consistent with the interpretation that TRBP inhibits the interplay of Tax with the transcription machinery or accessory factors.

Identification of pirin, a novel highly conserved nuclear protein.

In this article we describe the molecular cloning of Pirin, a novel highly conserved 32-kDa protein consisting of 290 amino acids. Pirin was isolated by a yeast two-hybrid screen as an interactor of nuclear factor I/CCAAT box transcription factor (NFI/CTF1), which is known to stimulate adenovirus DNA replication and RNA polymerase II-driven transcription. Pirin mRNA is expressed weakly in all human tissues tested. About 15% of all Pirin cDNAs contain a short 34-base pair insertion in their 5'-untranslated regions, indicative of alternative splicing processes. Multiple Pirin transcripts are expressed in skeletal muscle and heart. Western blots and immunoprecipitations employing monoclonal anti-Pirin antibodies reveal that Pirin is a nuclear protein. Moreover, confocal immunofluorescence experiments demonstrate a predominant localization of Pirin within dot-like subnuclear structures. Homology searches using the BLAST algorithm indicate the existence of Pirin homologues in mouse and rat. The N-terminal half of Pirin is significantly conserved between mammals, plants, fungi, and even prokaryotic organisms. Genomic Southern and Western blots demonstrate the presence of Pirin genes and their expression, respectively, in all mammalian cell lines tested. The expression pattern, the concentrated localization in subnuclear structures, and its interaction with NFI/CTF1 in the two-hybrid system classify Pirin as a putative NFI/CTF1 cofactor, which might help to gain new insights in NFI/CTF1 functions.

HDF2, the second subunit of the Ku homologue from Saccharomyces cerevisiae.

The high affinity DNA binding factor (HDF) protein of Saccharomyces cerevisiae is composed of two subunits and specifically binds ends of double-stranded DNA. The 70-kDa subunit, HDF1, shows significant homology with the 70-kDa subunit of the human Ku protein. Like the Ku protein, HDF1 has been shown to be involved in recombination and double stranded DNA break repair. We have purified and cloned HDF2, the second subunit of the HDF protein. The amino acid sequence of HDF2 shows a 45.6% homology with the 80-kDa subunit of the Ku protein. HDF1 by itself does not bind DNA, while HDF2 protein on its own seems to displays DNA binding activity. Targeted disruption of the HDF2 gene causes a temperature-sensitive phenotype for growth comparable to the phenotype of hdf1(-) strains. The human Ku protein cannot complement this temperature-sensitive phenotype. hdf2(-) strains are sensitive to bleomycin and methyl methanesulfonate, but this sensitivity is reduced in comparison with hdf1(-) strains.

Characterization of the single-strand-specific BPV-1 origin binding protein, SPSF I, as the HeLa Pur alpha factor.

SPSF I and II are two cellular proteins which bind specifically to single-stranded DNA. SPSF I and II binding sites are found in the minimal origin of replication of BPV-1 DNA and near the P2 promoter of the cellular c-myc gene. DNA-binding properties of the two proteins to single-stranded oligonucleotides of different lengths and sequences were quantified by determination of DNA-binding constants. The binding constant of SPSF proteins to the lower strand of the BPV-1 origin was determined to be 1.5 x 10(-10) M-1. Peptide sequences derived from purified SPSF I and II revealed the identity of at least one of the SPSF proteins with the so-called HeLa Pur alpha factor. The HeLa Pur alpha factor was identified previously by virtue of its capacity to bind to purine-rich strands of the PUR element found in initiation zones of DNA replication [Bergemann, A.D., Ma,Z.-W. and Johnson, E.M. (1992) Mol. Cell. Biol. 12, 5673-5682]. Expression of the Pur cDNA confirmed the identity of the Pur alpha protein with the 42 kDa SPSF I protein. Analysis of several Pur alpha cDNA clones revealed the existence of an extended 3'-untranslated region in all Pur mRNAs.

Prion protein PrPc interacts with molecular chaperones of the Hsp60 family.

Prions mediate the pathogenesis of certain neurodegenerative diseases, including bovine spongiform encephalopathy in cattle and Creutzfeldt-Jakob disease in humans. The prion particle consists mainly, if not entirely, of PrPSc, a posttranslationally modified isoform of the cellular host-encoded prion protein (PrPc). It has been suggested that additional cellular factors might be involved in the physiological function of PrPc and in the propagation of PrPSc. Here we employ a Saccharomyces cerevisiae two-hybrid screen to search for proteins which interact specifically with the Syrian golden hamster prion protein. Screening of a HeLa cDNA library identified heat shock protein 60 (Hsp60), a cellular chaperone as a major interactor for PrPc. The specificity of the interaction was confirmed in vitro for the recombinant proteins PrPc23-231 and rPrP27-30 fused to glutathione S-transferase with recombinant human Hsp60 as well as the bacterial GroEL. The interaction site for recombinant Hsp60 and GroEL proteins was mapped between amino acids 180 and 210 of the prion protein by screening with a set of recombinant PrPc fragments. The binding of Hsp60 and GroEL occurs within a region which contains parts of the putative alpha-helical domains H3 and H4 of the prion protein.

CTF5--a new transcriptional activator of the NFI/CTF family.

NFI/CTF is a family of polypeptides involved in stimulating the initiation of adenovirus DNA replication and the activation of transcription driven by RNA polymerase II. Several naturally occurring NFI/CTF variants display distinctive transactivation activities in vivo. To define more precisely the role of the NFI/CTF family in regulating gene expression, we cloned the splice variant CTF5, analyzed transcriptional activation patterns in a yeast transcription assay, and compared it with other CTF proteins. CTF5, which lacks exons 9 and 10 including a CTD-like motif essential for transcriptional activation by full-length CTF1, enhances transcription to a greater extent than CTF1. In addition, CTF5 is even more active than CTF7, which lacks exons 7-9. These findings indicate that CTF proteins formed by differential splicing display a much broader range of transcriptional activities as observed previously.

Involvement of the Saccharomyces cerevisiae HDF1 gene in DNA double-strand break repair and recombination.

The HDF1 protein of Saccharomyces cerevisiae shares biochemical properties and structural homology with the 70-kDa subunit of the human autoantigen Ku. The Ku protein, a heterodimer composed of a 70-kDa subunit and an 80-kDa subunit, has been identified as the regulatory subunit of the DNA-dependent protein kinase. This enzyme has recently been shown to be involved in DNA repair and recombination processes in mammalian cells. Here we show that hdf1-disrupted S. cerevisiae strains are strongly sensitive toward the radiomimetic antibiotic bleomycin. In addition, mating-type switching and rates of spontaneous mitotic recombination are strongly reduced. This phenotype is similar to that of mammalian cells lacking components of the DNA-dependent protein kinase holoenzyme, suggesting that HDF1 participates in and exerts equivalent functions in S. cerevisiae.

Cloning, sequence determination and functional expression of the genes encoding adenovirus type-4 polymerase and the terminal protein precursor.

Sequences of the open reading frames encoding adenovirus type 4 (Ad4) DNA polymerase and the terminal protein precursor were determined. Sequence comparisons with the corresponding genes and proteins from Ad2 and Ad5 show high overall identity, but significant differences in those portions of the two proteins thought to be essential for their biological activities. Both Ad4 proteins were functionally expressed in insect cells from the corresponding cDNAs.

Recombinant prion protein rPrP27-30 from Syrian golden hamster reveals proteinase K sensitivity.

PrP27-30 represents the protease-resistant core of the prion protein and was found to be the main component in Scrapie prion preparations. Recombinant (r) PrP27-30 corresponding to aa 90-231 from the Syrian golden hamster prion protein was expressed as a fusion with GST in E. coli and secreted from insect cells infected with recombinant baculoviruses, GST::rPrP27-30 isolated from either system was purified to homogenity by glutathione-Sepharose chromatography. rPrP27-30 from both systems was generated by direct cleavage of GST::rPrP27-30 in the presence of thrombin revealing a molecular weight of 17 kDa. GST::rPrP27-30 as well as the authentic protein rPrP27-30 were identified by immunoblotting employing a polyclonal antibody directed against a peptide corresponding to aa 95-110 of the Syrian golden hamster prion protein. In contrast to scrapie prior PrP27-30, the recombinant proteins GST::rPrP27-30 and rPrP27-30 were both sensitive towards proteinase K, suggesting that the molecules lack infectivity.

Overexpression of active Syrian golden hamster prion protein PrPc as a glutathione S-transferase fusion in heterologous systems.

This article describes a procedure which permits for the first time the isolation of the prion protein PrPc from the Syrian golden hamster in heterologous systems. Using a glutathione S-transferase (GST) fusion approach, milligram amounts of stable, soluble, and homogeneous GST::PrPc protein were obtained in Escherichia coli and with baculovirus-infected insect cells. Authentic PrPc was released from the immobilized fusion protein by direct cleavage with thrombin. GST::PrPc expressed in these two expression systems and also authentic PrPc released by thrombin cleavage were recognized by a polyclonal antibody directed against amino acid 95 to 110 of the golden hamster PrPc protein. GST::PrPc was not detected by a monoclonal antibody recognizing the region encompassing amino acids 138 to 152 of the human prion protein. The fusion protein was sensitive to proteinase K digestion, demonstrating that the cellular rather than the proteinase K-resistant scrapie isoform was produced.

Organization of the alpha-globin promoter and possible role of nuclear factor I in an alpha-globin-inducible and a noninducible cell line.

Nuclear factor I (NFI) was suggested to be involved in the expression of the human alpha-globin gene. Two established cell lines, which express alpha-globin differentially, were therefore compared for differences in binding of NFI at the alpha-globin promoter in vivo. HeLa cells, in which alpha-globin is repressed, show a high density promoter occupation with several proteins associated with structurally distorted DNA. Cell line K562, which is inducible for alpha-globin, surprisingly was found to be heterogeneous consisting mainly of cells (approximately 95%) unable to express alpha-globin. However, the promoter of the nonexpressing K562 cells was clearly different from that of HeLa cells, being occupied only at basal transcriptional elements. Therefore, the alpha-globin gene in these K562 cells may not be truly repressed, but in an intermediate state between repression and active transcription. The NFI site of the alpha-globin promoter appeared occupied in HeLa but free of proteins in K562 cells. All cells of both cell lines produce NFI, but the composition and DNA binding affinity of NFI species differ significantly between the two cell lines. Therefore, distinct forms of NFI may repress alpha-globin transcription in HeLa cells. However, NFI is apparently not involved in establishing the latent transcriptional state of the majority of K562 cells.