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Polyclonal antisera to either a synthetic OipA peptide or a recombinant OipA protein detected OipA expression in Helicobacter pylori and correlated with functional oipA status determined by PCR sequence (sensitivity and specificity of >94%). Immunoblotting is a simple and accurate method for detecting expression of the important virulence factor OipA.
The generation of T, B and NK lymphocyte lineages from pluripotent hematopoietic stem cells is dependent upon the early expression of the Ikaros locus which by means of alternative splicing produces a variety of zinc finger DNA binding transcription factors. We assessed the general biological importance of Ikaros by studying its conservation and expression in teleost fish and amphibians. Portions of Ikaros cDNA from rainbow trout and Xenopus were amplified by reverse transcription-polymerase chain reaction (RT-PCR). They show roughly 75% conservation of the amino acid sequence with mammalian Ikaros. The trout fragment was then used to isolate full-length Ikaros clones from a trout thymocyte cDNA library. In mice and humans, Ikaros produces six alternatively spliced isoforms, but in trout two additional novel splice variants designated Ik-7 and Ik-8 were also found. Ik-7 is expressed in a similar fashion to Ik-1 and Ik-2, the predominant isoforms expressed in mammalian lymphocytes. In trout and zebrafish, as in mammals, Ikaros is a single-copy gene, but in Xenopus segregation analysis demonstrates that Ikaros has been duplicated, most likely a result of polyploidization. We then examined the expression of Ikaros in trout and Xenopus tumor T cell lines via Northern blot, RT-PCR, immunofluorescence and Western blot analysis. Overall, Ikaros is expressed in a lymphoid-specific fashion similar to that found in mice and humans. In addition Ikaros is expressed early in trout ontogeny, beginning roughly at days 3-4 in the yolk sac and at day 5-6 in the embryo proper. The conservation of Ikaros structure and expression confirms it as a master switch of hematopoiesis.
During embryogenesis in the sea urchin Strongylocentrotus purpuratus, there is a shift from one histone mRNA population to another. The early and late embryonic histone mRNAs, previously shown to differ considerably in sequence from each other by hybrid melting studies, are shown here to differ also in electrophoretic mobility on polyacrylamide gels as the positions of the early and late mRNAs are completely noncoincident. The various species of both early and late samples are identified as particular histone mRNAs by hybridization to cloned histone DNAs containing part of the early-type repeat unit or to restriction enzyme fragments derived from these unit. Four bands in the early mRNA sample are identified as H1, H3, H2A " H2B, and H4 mRNA while at least 10 bands can be seen in the late mRNA preparation with unambiguous identification of H1, H2B, and H4 mRNAs. A cluster of late species is shown to contain both H3 and H2A mRNA. When a polysomal RNA preparation from the 26-h embryo is hybridized to the histone DNA, eluted, and then translated in vitro in a wheat germ system, the histone products migrate in the position of late histones when subjected to electrophoresis on Triton X-urea gels. Using DNA which contains genes for H2A + H3 or H2A alone, we demonstrate the specificity of the early-type DNA probes for these two late histones. Therefore, by hybridization of newly synthesized RNAs and translation of the total polysomal RNA present in the late embryo, it is shown that mRNAs for all five histone classes may cross-react with the cloned early-type DNA. The hybrids formed, however, are much less stable than those formed with the early histone mRNA. In vitro translation of total cytoplasmic RNA from various embryonic stages indicates that transition between the two classes occurs during most of the blastula period.
In this work, the genes that encode the FliM and FliN proteins of Rhodobacter sphaeroides were characterized. These genes are part of a large flagellar gene cluster in which six additional open reading frames encoding products homologous to FliL, FliO, FliP, FliQ, FliR, and FlhB proteins from other bacteria were identified. The inactivation of the fliM gene gave a nonflagellate phenotype (Fla-), suggesting that FliM is required for flagellar assembly. Complementation analysis of this fliM mutant indicated that fliM and fliN transcription starts beyond the 5' end of fliK and terminates after fliN.
The product of the Epstein-Barr virus BZLF1 gene encodes a protein which is related to c-fos, it has been shown to bind specifically to a consensus AP-1 site, and its expression in latently Epstein-Barr virus-infected lymphocytes is sufficient to trigger the viral lytic cycle. We identified several elements within the BZLF1 promoter (Zp) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. These elements fall into two classes based on the factors which bind to these sequences and their resulting functional behavior. Four of the elements are homologous (ZI elements) and share homology to a protein-binding domain in the promoter region of the coordinately expressed BRLF1 gene. When cloned upstream of heterologous promoters, the ZI elements function as silencers which exhibit TPA-inducible enhancer activity. A distinct TPA-responsive element (ZII) is located near the TATA box and shares homology with the AP-1-binding site in the c-jun promoter. A synthetic oligonucleotide with a sequence corresponding to the ZII element effectively competes for binding of nuclear factors to the c-jun AP-1 site. Furthermore, we found that a complex of c-jun and c-fos bound to the ZII domain.
Expression of the Epstein-Barr virus (EBV) BZLF1 gene in latently infected lymphocytes is sufficient to trigger the viral lytic cycle. As shown in the accompanying report (E. Flemington and S.H. Speck, J. Virol. 64:1217-1226, 1990), the promoter for the BZLF1 gene (Zp) contains two distinct types of elements (ZI and ZII [an AP-1-like domain]) which are responsive to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA), an inducer of the viral lytic cycle. Although Zp can be activated with TPA in an EBV-negative Burkitt's lymphoma cell line (Ramos), its activity is considerably lower than in EBV-positive cell lines which can be induced with TPA. Here we show that the protein product of the BZLF1 gene (ZEBRA) can transactivate its own promoter by a mechanism which involves direct binding to a region distinct from the ZI and ZII element. Moreover, we show that this region is composed of two distinct ZEBRA-binding-transactivation domains. Interestingly, these two domains are not homologous, and while one domain (ZIIIA) is similar to previously described ZEBRA-binding domains, the second (ZIIIB) is a higher-affinity site which bears no detectable homology to the consensus ZEBRA recognition sequence. We also show that transactivation is independent of the otherwise essential ZII domain, suggesting that ZEBRA binding may functionally replace or supercede the need for a functional ZII domain. This observation supports a model for activation of the lytic cycle whereby synthesis of a critical level of ZEBRA signals commitment to BZLF1 transcription and initiation of the lytic cascade.
In patients with localized prostate cancer, radical prostatectomy and radiation therapy, although effective in controlling localized disease, are often associated with significant side effects attributable to injury of adjacent tissues. Moreover, patients with metastatic disease eventually fail systemic hormonal or chemotherapy because of the development of progressive, refractory disease. In this study, we evaluated the safety and efficacy of a novel suicide gene therapy that could potentially spare normal tissue while bypassing molecular mechanisms of apoptosis resistance by using chemically inducible effector caspases to trigger apoptosis in prostate cancer cells. Initially, we compared the ability of a panel of inducible Fas signaling intermediates to kill human and murine prostate cancer cell lines. On the basis of the superior killing by downstream caspase-1 and caspase-3, replication-deficient adenoviral vectors expressing conditional caspase-1 (Ad-G/iCasp1) or caspase-3 (Ad-G/iCasp3), regulated by nontoxic, lipid-permeable, chemical inducers of dimerization (CID), were constructed. Upon vector transduction followed by CID administration, aggregation and activation of these recombinant caspases occur, leading to rapid apoptosis. In vitro, both human (LNCaP and PC-3) and murine (TRAMP-C2 and TRAMP-C2G) prostate cancer cell lines were efficiently transduced and killed in a CID-dependent fashion. In vivo, direct injection of Ad-G/iCasp1 into s.c. TRAMP-C2 tumors caused focal but extensive apoptosis without evidence for a bystander effect at the maximal viral dose (i.e., 2.5 x 10(10) viral particles/25 microl) in host animals that also received CID compared with control animals. Treatment with Ad-G/iCasp1 plus CID resulted in a transient, yet significant, reduction both in tumor growth and volume compared with tumors treated with vector but not CID (P < 0.035) or vector-diluent plus CID (P < 0.022), both of which grew more rapidly. These results demonstrate that CID-regulated, caspase-based suicide gene therapy is safe and can inhibit the growth of experimental prostate cancer in vitro and in vivo through potent induction of apoptosis, providing a rationale for further development.
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The putative G0/G1 switch regulatory gene G0S30/EGR1/NFGI-A show increased expression shortly after adding concanavalin-A (ConA) to cultured T lymphocytes. However, it is reported that lymphocytes from mice in which the gene has been deleted proliferate normally in response to ConA. This suggests that G0S30 expression is not critical for the response. Paradoxically, others report that proliferation of ConA-stimulated rat lymphocytes is inhibited by an antisense oligonucleotide complementary to G0S30. Because the G0S30 sequence is highly conserved between species, we used a similar oligonucleotide (differing by 1 base) to show for humans that the response to ConA is also inhibited. However, no oligonucleotide-induced changes in the concentrations of G0S30 protein or mRNA are detectable. This suggests that the oligonucleotide is not acting by influencing the expression of G0S30, and may be targeting another gene. The phosphorothioated oligonucleotide was maximally inhibitory at a 50 nM concentration, which is near to the "physiological" concentration found with CpG-containing oligonucleotides to activate mouse B lymphocytes. In the present work, increasing the concentration above 50 nM, or adding further quantities of control oligonucleotides, decreased the inhibition. It is suggested that by using low oligonucleotide concentrations (the "stealth" approach), one may avoid "tripping" an endogenous defense system directed against exogenous oligonucleotides, yet still get sufficient uptake to inhibit lymphocyte activation.
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The Epstein-Barr virus (EBV) lytic switch gene, BZLF1, is tightly regulated in latently infected B cells. The BZLF1 gene promoter (Zp) contains several cis elements that have been previously shown to respond to inducers of the viral lytic cycle. These include four copies of an element referred to as the ZI domains and an element that contains a consensus CRE/AP-1 motif (ZII domain). In addition, Zp is autoregulated through two sites that bind the BZLF1 gene product Zta. The ZI domains have been shown to bind the ubiquitous cellular transcription factors Sp1 and Sp3 and/or the myocyte enhancer factor 2D (Liu et al., EMBO J. 16:143-153, 1997; Liu et al., Virology 228:9-16, 1997). Here we present a functional analysis of the ZII domain and show: (i) ATF-1 and ATF-2 appear to be the predominant cellular factors that bind to the CRE/AP-1 motif present in the ZII domain; and (ii) the region immediately upstream of the CRE/AP-1 motif contains a potent negative cis element, mutation of which results in a >10-fold increase in Zp activity. The negative cis element (ZIIR) in the ZII domain decreases both basal and induced Zp activity and thus is likely to play an important role in regulating reactivation of EBV. In addition, analysis of heterologous promoter constructs indicates that the function of ZIIR is context sensitive. Attempts to demonstrate a cellular factor binding to ZIIR have been unsuccessful, leaving unresolved the mechanism by which repression is mediated.
We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.
We examined restriction fragment length polymorphisms (RFLPs) of the switch region genes of the IgM (S mu) and IgA1 (S alpha 1) heavy chain in 78 Japanese children with IgA nephropathy and 88 normal Japanese controls. Genomic DNA obtained from patients and controls was digested with the restriction endonuclease SacI, transferred to nylon membrane using Southern blot procedure, and hybridized with a DNA probe homologous to S mu. This probe detects RFLPs at the S mu and S alpha 1 loci by enhanced chemiluminescence. The genotypic frequency of the S mu and S alpha 1 alleles in patients with IgA nephropathy was similar to normal controls. However, there was a significant association of genotypes with the pathological severity. There was a decreased frequency of the 2.6/2.1 kb S mu heterozygous genotype in patients showing diffuse mesangial proliferation compared to controls or patients showing minimal or focal mesangial proliferation. Our results suggest that immunoglobulin heavy chain switch region genes may not influence susceptibility to IgA nephropathy in children, but may influence the pathological expression of childhood IgA nephropathy.
It is feasible to restrict transgene expression to a tissue or region in need of therapy by using promoters that respond to focusable physical stimuli. The most extensively investigated promoters of this type are radiation-inducible promoters and heat shock protein gene promoters that can be activated by directed, transient heat. Temporal regulation of transgenes can be achieved by various two- or three-component gene switches that are triggered by an appropriate small molecule inducer. The most commonly considered gene switches that are reviewed herein are based on small molecule-responsive transactivators derived from bacterial tetracycline repressor, insect or mammalian steroid receptors, or mammalian FKBP12/FRAP. A new generation of gene switches combines a heat shock protein gene promoter and a small molecule-responsive gene switch and can provide for both spatial and temporal regulation of transgene activity.
We report the sequence of 3978 bp of the Agrobacterium tumefaciens chromosome which contains a putative operon encoding the homologues of the transmembrane proton channel protein MotA, and the flagellar switch proteins FliM, FliN and FliG. Two transposon insertions in fliG result in a non-flagellate phenotype, indicating that this gene at least is required for flagellar assembly.
Our studies using proteases to probe protein structure establish that binding to the upstream activating sequences (UASs) of two different yeast a-specific genes induces a conformational change in the pheromone/receptor transcription factor (PRTF), which is not observed upon binding to the UASs of either of two alpha-specific genes. We propose that this selective structural alteration exposes an activation region of PRTF when it binds a-specific genes, switching these genes on. The transcriptional activator MAT alpha 1 may activate alpha-specific genes by binding to the PRTF-alpha-specific UAS complex and unmasking the otherwise hidden activation surface of PRTF. We also show that the N-terminal third of PRTF is sufficient for specific DNA binding, while the middle third of the protein interacts with MAT alpha 1.
The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands.
Ecdysteroids are widely used as inducers for gene-switch systems based on insect ecdysteroid receptors and genes of interest placed under the control of ecdysteroid-response elements. We review here these systems, which are currently mainly used in vitro with cultured cells in order to analyse the role of a wide array of genes, but which are expected to represent the basis for future gene therapy strategies. Such developments raise several questions, which are addressed in detail. First, the metabolic fate of ecdysteroids in mammals, including humans, is only poorly known, and the rapid catabolism of ecdysteroids may impede their use as in vivo inducers. A second set of questions arose in fact much earlier with the pioneering "heterophylic" studies of Burdette in the early sixties on the pharmacological effects of ecdysteroids on mammals. These and subsequent studies showed a wide range of effects, most of them being beneficial for the organism (e.g. hypoglycaemic, hypocholesterolaemic, anabolic). These effects are reviewed and critically analysed, and some hypotheses are proposed to explain the putative mechanisms involved. All of these pharmacological effects have led to the development of a wide array of ecdysteroid-containing preparations, which are primarily used for their anabolic and/or "adaptogenic" properties on humans (or horses or dogs). In the same way, increasing numbers of patents have been deposited concerning various beneficial effects of ecdysteroids in many medical or cosmetic domains, which make ecdysteroids very attractive candidates for several practical uses. It may be questioned whether all these pharmacological actions are compatible with the development of ecdysteroid-inducible gene switches for gene therapy, and also if ecdysteroids should be classified among doping substances.