Functional interaction between cyclin T1/cdk9 and Puralpha determines the level of TNFalpha promoter activation by Tat in glial cells.

In addition to its stimulatory effect on transcription of the HIV-1 LTR, the early protein of HIV-1, Tat, exhibits detrimental effects on the CNS by deregulating the expression of several cytokines and immunomodulators including TNFalpha. Activation of the viral promoter by Tat requires several cellular proteins including cyclin T1 and its partner, cdk9, which upon association with the TAR sequence of the LTR, forms a complex that enhances the activity of RNA polymerase II. Here, we examined the involvement of cyclin T1/cdk9 in Tat-mediated transcriptional activation of the TNFalpha promoter which has no TAR sequence. Results from transfection of human astrocytic cells revealed that both cyclin T1 and cdk9 stimulate the basal promoter activity of TNFalpha, although the level of such activation is decreased in the presence of Tat. Ectopic expression of Puralpha, a brain-derived regulatory protein which binds to Tat, enhanced the basal level of TNFalpha transcription, yet exerted a negative effect on the level of Tat activation of the TNFalpha promoter. The antagonistic effect of Puralpha and Tat upon the TNFalpha promoter was diminished in the presence of cyclin T1 and cdk9, suggesting cooperativity of Puralpha with cyclin T1 and cdk9 in Tat activation of the TNFalpha promoter. Results from protein-protein binding studies showed the interaction of Puralpha with both cyclin T1 and cdk9 through distinct domains of Puralpha which are in juxtaposition with each other. Interestingly, the site for cyclin T1 binding within Puralpha is adjacent to the region which is important for Tat/Puralpha association. In light of these observations, we propose a model which ascribes a bridging role for Puralpha in assembling Tat, cyclin T1, and cdk9 around the promoter region of TAR-negative genes such as TNFalpha, which is responsive to Tat activation.

Growth inhibition of glioblastoma cells by human Pur(alpha).

Pur(alpha) is a multifunctional DNA- and RNA-binding protein implicated in a variety of biological events including transcription and replication. Further, this protein has the ability to form a complex with several cellular proteins which are important for cell proliferation including the transcription factor, E2F-1. Pur(alpha) has a modular structure highlighted by alternating three basic aromatic class I and two acidic leucine-rich class II repeats in the central region of the protein. Here, we demonstrate that ectopic overexpression of Pur(alpha) suppresses proliferation of a variety of transformed and tumor cells including human glioblastoma. By utilizing various deletion mutants of Pur(alpha) in colony formation assay, we identified the region spanning the first class II repeat (residues 107-131) and the second class I repeat (residues 148-170) of Pur(alpha) which participate in growth inhibitory action of Pur(alpha). Results from protein transduction experiments using the synthetic peptides representing residues 109-131 and 123-154 of Pur(alpha) in fusion with the arginine rich domain of HIV-1 Tat revealed cellular internalization and nuclear appearance of the Tat-Pur(alpha) fusion peptide after 2 h and its detection in nuclei up to 24 h after treatment. Glioblastoma cells treated with Tat-Pur(alpha) (109-131) and Tat-Pur(alpha) (123-154) exhibited 41 and 47% decrease, respectively, in proliferation. Altogether these results illustrate the efficacy of Pur(alpha) in suppressing glioblastoma cell growth and provide evidence for the potential use of this protein and its derivative(s) in blocking proliferation of tumor cells.

Single-stranded nucleic acid-binding protein, Pur alpha, interacts with RNA homologous to 18S ribosomal RNA and inhibits translation in vitro.

Pur alpha is a highly conserved, eukaryotic sequence-specific DNA- and RNA-binding protein involved in diverse cellular and viral functions including transcription, replication, and cell growth. Pur alpha exerts its activity in part by interacting with other viral and cellular proteins. One such protein is the human immunodeficiency virus (HIV) type I regulatory protein Tat. Earlier studies have demonstrated that this interaction is mediated by Pur alpha-associated RNA (PARNA) and that RNA immunopurified from mammalian expressed Pur alpha was capable of reconstituting the interaction between these two proteins. In the current study, we characterize four RNA species which were immunopurified with Pur alpha. Northern blot analysis with one of the PARNAs revealed a highly abundant signal of approximately 2.0 kilobases (kb) present in all cell lines tested. Sequence analysis of each of the four PARNA clones revealed a high homology to different regions of the human 18S ribosomal RNA sequence. Based on this homology, we investigated the influence of Pur alpha on translation. Luciferase assays were performed after coupled in vitro transcription/translation reactions with a vector containing a luciferase reporter construct and increasing concentrations of BSA, GST, and GST-Pur alpha. Inclusion of GST-Pur alpha in these reactions resulted in a dose-dependent inhibition of luciferase activity. Similar inhibition was observed with in vitro translation reactions performed with in vitro transcribed luciferase RNA and increasing concentrations of GST-Pur alpha. In control experiments, inclusion of increasing concentrations of GST-Pur alpha with luciferase protein resulted in no effect on luciferase activity. Taken together, these data demonstrate that Pur alpha inhibits translation reactions in vitro. Moreover, this Pur alpha-mediated inhibition of translation can be abrogated by HIV-1 Tat protein.

Helix-destabilizing properties of the human single-stranded DNA- and RNA-binding protein Puralpha.

Puralpha is a ubiquitous nucleic acid-binding protein which has been implicated in the control of eukaryotic gene transcription. Further, Puralpha associates with DNA sequences positioned in close proximity to viral and cellular origins of replication suggesting a role for this protein in DNA replication. As initiation of transcription and replication require alteration in the structure of duplex DNA, we investigated the DNA unwinding activity of this single-stranded nucleic acid-binding protein. Here we demonstrate that Puralpha has the ability to displace an oligonucleotide annealed to single-stranded M13 DNA. The helix unwinding activity of Puralpha was dose-, time- and temperature-dependent and ATP-independent. Results from mapping studies revealed that the central region of Puralpha, spanning amino acids 72-274, was important for the helix-destabilizing activity of this protein. The region of Puralpha which was involved in the helix-destabilizing activity mapped to the DNA-binding domain of this protein. Results from heat inactivation experiments demonstrated that the helix-destabilizing activity of Puralpha correlates with its capacity to interact with DNA containing the PUR element. Taken together, these studies demonstrate that Puralpha possesses helix-destabilizing activity and that this activity maps to and correlates with its ability to interact with DNA.

Association of Pur alpha and E2F-1 suppresses transcriptional activity of E2F-1.

Protein-protein interaction can play an important role in the control of several biological events including gene transcription, replication and cell proliferation. E2F-1 is a DNA-binding transcription factor which, upon interaction with its target DNA sequence, induces expression of several S phase specific genes allowing progression of the cell cycle. Evidently, the activity of this protein is modulated by its cellular partner, pRb, which in the hypophosphorylated form, binds to E2F-1 and inactivates its transcriptional ability. In this study, we have demonstrated that expression of a sequence-specific single-stranded DNA binding protein, Pur alpha, in cells decreases the ability of E2F-1 to exert its transcriptional activity upon the responsive promoter derived from DHFR. Results from band shift experiments revealed that while Pur alpha does not recognize the double-stranded DNA fragment containing the E2F-1 binding site, it has the ability to inhibit E2F-1 interaction with its target DNA sequence. Results from GST pull-down assays and the combined immunoprecipitation/Western blot analysis of nuclear extracts revealed a direct association of E2F-1 with Pur alpha in the absence of the DNA molecule containing the E2F-1 binding site. The association of Pur alpha with E2F-1 may increase the stability of E2F-1, as a higher level of E2F-1 was detected in cells coexpressing Pur alpha and E2F-1. The importance of these observations with respect to the role of Pur alpha in the control of cell cycle progression is discussed.

Association of HIV-1 Tat with the cellular protein, Puralpha, is mediated by RNA.

The interaction between two regulatory proteins plays a crucial role in the control of several biological events, including gene transcription. In this report, we demonstrate that the interaction between the cellular sequence-specific single-stranded DNA binding protein Puralpha and the HIV type 1 (HIV-1) Tat protein is mediated by specific ribonucleic acids. The region of Tat that is important for its interaction with Puralpha includes the region demonstrated to bind Tat's viral RNA target, TAR. A 10-nucleotide GC-rich consensus sequence identified in RNAs associated with Puralpha derived from human U-87MG cells plays an important role in the Puralpha:Tat interaction as examined by an in vitro reconstitution assay. Furthermore, expression of the Puralpha-associated RNA in these cells enhances transcriptional activation of the HIV-1 promoter by Tat and Puralpha.

Self-association of Puralpha is mediated by RNA.

The 322 amino acid cellular protein, Puralpha, is a sequence-specific single-stranded DNA-binding protein implicated in control of transcription and replication. Previous studies have demonstrated that the interaction between Puralpha and its target DNA sequence results in the formation of multimeric complexes. In this study, we demonstrate that Puralpha can self-associate in the absence of DNA. This self-association, while independent of DNA, is mediated by RNA. Through in vitro studies with bacterially expressed glutathione S-transferase fusion proteins, and the synthetic peptides corresponding to various central regions of Puralpha, the domain which is important for the self-association of Puralpha is localized to acidic leucine-rich repeats. Interestingly, these repeats have previously been shown to interact with the human immunodeficiency virus 1 (HIV-1) Tat protein and in this study we demonstrate that Tat is able to disrupt the self- association of Puralpha. We have recently cloned a Puralpha associated-RNA, PU-RNA, and here we show that PU-RNA can specifically reconstitute the self-association of Puralpha. RNA not only mediates the self-association of Puralpha, but also modulates the ability of Puralpha to interact with its target DNA sequence. Electrophoretic mobility shift assays performed with and without RNase treatment demonstrate that RNA inhibits the interaction between Puralpha and its target DNA sequence. Moreover, we demonstrate that the self-association of Puralpha can be reconstituted by a specific oligonucleotide encompassing the Puralpha binding site. The implications of these findings with respect to Puralpha's role in transcription and replication are discussed.

Role of the Agrobacterium tumefaciens VirD2 protein in T-DNA transfer and integration.

VirD2 is one of the key Agrobacterium tumefaciens proteins involved in T-DNA processing and transfer. In addition to its endonuclease domain, VirD2 contains a bipartite C-terminal nuclear localization sequence (NLS) and a conserved region called omega that is important for virulence. Previous results from our laboratory indicated that the C-terminal, bipartite NLS and the omega region are not essential for nuclear uptake of T-DNA, and further suggested that the omega domain may be required for efficient integration of T-DNA into the plant genome. In this study, we took two approaches to investigate the importance of the omega domain in T-DNA integration. Using the first approach, we constructed a T-DNA binary vector containing a promoterless gusA-intron gene just inside the right T-DNA border. The expression of beta-glucuronidase (GUS) activity in plant cells transformed by this T-DNA would indicate that the T-DNA integrated downstream of a plant promoter. Approximately 0.4% of the tobacco cell clusters infected by a wild-type A. tumefaciens strain harboring this vector stained blue with 5-bromo-4-chloro-3-indolyl beta-D-glucuronic acid (X-gluc). However, using an omega-mutant A. tumefaciens strain harboring the same binary vector, we did not detect any blue staining. Using the second approach, we directly demonstrated that more T-DNA is integrated into high-molecular-weight plant DNA after infection of Arabidopsis thaliana cells with a wild-type A. tumefaciens strain than with a strain containing a VirD2 omega deletion/substitution. Taken together, these data indicate that the VirD2 omega domain is important for efficient T-DNA integration. To determine whether the use of the T-DNA right border is altered in those few tumors generated by A. tumefaciens strains harboring the omega mutation, we analyzed DNA extracted from these tumors. Our data indicate that the right border was used to integrate the T-DNA in a similar manner regardless of whether the VirD2 protein encoded by the inciting A. tumefaciens was wild-type or contained an omega mutation. In addition, a mutant VirD2 protein lacking the omega domain was as least as active in cleaving a T-DNA border in vitro as was the wild-type protein. Finally, we investigated the role of various amino acids of the omega and bipartite NLS domains in the targeting of a GUS-VirD2 fusion protein to the nucleus of electroporated tobacco protoplasts. Deletion of the omega domain, or mutation of the 10-amino-acid region between the two components of the bipartite NLS, had little effect upon the nuclear targeting of the GUS-VirD2 fusion protein. Mutation of both components of the NLS reduced, but did not eliminate, targeting of the fusion protein to the nucleus.

The use of a thermostable beta-glucanase gene from Clostridium thermocellum as a reporter gene in plants.

In order to take advantage of the high thermostability of its product, beta-1,3;1,4-glucanase (lichenase), we used a modified version of the licB gene from Clostridium thermocellum as a reporter gene for the analysis of gene expression in transformed plants. The coding region of the licB gene was truncated at both ends. The truncated enzyme retained its activity and thermostability. The modified gene (m-licB), with and without a plant leader peptide-encoding sequence, was expressed in tobacco plants under control of either the Agrobacterium octopine TR-DNA 2' gene promoter or the promoter of the gene for the small subunit of ribulose-1,5-bisphosphate carboxylase. Expression of licB can be measured quantitatively and accurately, the assay is sensitive and simple enough to be used for analysis of various gene fusion systems or for screening of transformants. The enzyme is very stable and remains active in tissue extracts even after storage for 1 year and survives many thawing-freezing cycles. The lichenase-encoding gene was expressed at high levels in transformed tobacco plants without any apparent detrimental effects on vegetative growth or flowering.

Expression of a bacterial endo beta-1,3-glucanase gene under the control of CAMV 35S promoter in Nicotiana plumbaguinifolia protoplasts.

A gene which codes for a thermostable endo beta-1,3-glucanase (EC 3.2.1.39) from a Gram positive anaerobic thermophilic bacteria Clostridium thermocellum F7, was fused to 35S promoter and polyadenylation signal of Cauliflower Mosaic Virus (CaMV) strain Cabb B-D. This chimaeric gene fusion was introduced into Nicotiana plumbaguinifolia protoplasts using PEG-mediated DNA transfer method of transformation. Transient expression of the thermostable endo beta-1,3-glucanase was carried out in the protoplasts and was assayed at 70 degrees C pH 8.0, suggesting that the chimaeric gene fusion: (i) is correctly transcribed and translated in plant cells; (ii) the product of translation (the thermostable endo beta-1,3-glucanase protein) is easy to assay since most of the plants' enzymes have their optimal reaction temperature at 40-60 degrees C and at neutral or weak-acidic condition which is a characteristic of plant cells; (iii) can be used as a model for studying and understanding some of the mechanisms of plant defence systems at the enzyme protein level, in case of stress conditions.

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. I. Construction of vector plasmids for transfer into plants and expression of a modified gene for beta-1,3-glucanase from Clostridium thermocellum in tobacco protoplasts]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. I. Konstruirovanie vektorynykh plazmid dlia perenosa v rasteniia i ékspressiia modifitsirovannogo gena beta-1,3-gliukanazy iz Clostridium thermocellum v protoplastakh tabaka.

We constructed two vectors, pC27-glc and pC29-glc, that allow expression of the beta-1,3-glucanase gene (glc) in plant cells. The glc gene was previously cloned from anaerobic thermophilous bacterium Clostridium thermocellum. To increase the efficiency of expression, the N-terminal fragment of the glc gene encoding bacterial transient peptide was deleted, and hybrid variants of lacZ-glc were obtained. Analysis of expression of the hybrid genes in Escherichia coli showed that deletion of the fragment corresponding to 31 amino acids (a.a.) of beta-glucanase affected neither activity nor thermostability of the enzyme. The modified gene was subcloned into two vectors, pC27 and pC29, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter from Arabidopsis, respectively. Each of the resulting plasmids, pC27-glc and pC29-glc, was transfected into protoplasts of Nicotiana plumbaginifolia. Both the plasmids were shown to allow a high level of activity of the thermostable beta-1,3-glucanase. We plan to use the vectors obtained for transformation of agrobacteria and construction of transgenic plants.

[Construction and analysis of transgenic plants of Nicotiana tabacum L. expressing a bacterial gene for beta-1,3-glucanase. II. Transgenic tobacco plants expressing the bacterial beta-glucanase gene from Clostridium thermocellum,--a model for studying the differential expression of stress response-related genes]. / Konstruirovanie i analiz transgennykh rastenii Nicotiana tabacum L. s ékspressiruiushchimsia bakterial'nym genom beta-1,3-gliukanazy. II. Transgennye rasteniia tabaka, ékspressiruiushchie bakterial'nye gen beta-gliukanazy iz Clostridium thermocellum,--model' dlia izucheniia differentsial'noi ékspressii genov stressovogo otveta.

The modified hybrid beta-1,3-glucanase gene (glc) of Clostridium thermocellum was expressed in tobacco Nicotiana tabacum. The glc gene was cloned into two plasmids, pC27-glc and pC29-glc, in which its expression was controlled by the TR2' promoter of the 2' gene of T-DNA and the rbcS promoter of Arabidopsis, respectively. These constructions were used for transformation of agrobacteria followed by transfer into plants. In transformed plants, each plasmid caused a high level of activity of thermostable bacterial glucanase not observed in reference plants. The plants obtained were used to study activation of some defense-related genes induced by their interaction with either tobacco mosaic virus (TMV) or a pathogenic fungus.