Mobilan: a recombinant adenovirus carrying Toll-like receptor 5 self-activating cassette for cancer immunotherapy.

Toll-like receptor 5 (TLR5) is considered an attractive target for anticancer immunotherapy. TLR5 agonists, bacterial flagellin and engineered flagellin derivatives, have been shown to have potent antitumor and metastasis-suppressive effects in multiple animal models and to be safe in both animals and humans. Anticancer efficacy of TLR5 agonists stems from TLR5-dependent activation of nuclear factor-κB (NF-κB) that mediates innate and adaptive antitumor immune responses. To extend application of TLR5-targeted anticancer immunotherapy to tumors that do not naturally express TLR5, we created an adenovirus-based vector for intratumor delivery, named Mobilan that drives expression of self-activating TLR5 signaling cassette comprising of human TLR5 and a secreted derivative of Salmonella flagellin structurally analogous to a clinical stage TLR5 agonist, entolimod. Co-expression of TLR5 receptor and agonist in Mobilan-infected cells established an autocrine/paracrine TLR5 signaling loop resulting in constitutive activation of NF-κB both in vitro and in vivo. Injection of Mobilan into primary tumors of the prostate cancer-prone transgenic adenocarcinoma of the mouse prostate (TRAMP) mice resulted in a strong induction of multiple genes involved in inflammatory responses and mobilization of innate immune cells into the tumors including neutrophils and NK cells and suppressed tumor progression. Intratumoral injection of Mobilan into subcutaneously growing syngeneic prostate tumors in immunocompetent hosts improved animal survival after surgical resection of the tumors, by suppression of tumor metastasis. In addition, vaccination of mice with irradiated Mobilan-transduced prostate tumor cells protected mice against subsequent tumor challenge. These results provide proof-of-concept for Mobilan as a tool for antitumor vaccination that directs TLR5-mediated immune response toward cancer cells and does not require identification of tumor antigens.

Modified ELISA for the detection of neomycin phosphotransferase II in transformed plant species.

Identifying transformed plant lines carrying the antibiotic resistance marker gene, neomycin phosphotransferase II, requires a more definitive test than the ability of the plant to grow on kanamycin. Although a number of alternative assays have been described, most are cumbersome, time consuming and/or require the use of radioisotopes. This report describes an ELISA for the detection of the neomycin phosphotransferase II enzyme in transformed plant tissue. The ELISA utilises commercially available antibodies and provides a number of advantages, including an extremely low background, a reduction in the amount of tissue required for testing, and semi-quantitative data on neomycin phosphotransferase II gene expression. This method has been applied successfully to a number of independently transformed lines in nine plant species.

Controlled cytokinin production in transgenic tobacco using a copper-inducible promoter

The cytokinin group of plant hormones regulates aspects of plant growth and development, including the release of lateral buds from apical dominance and the delay of senescence. In this work the native promoter of a cytokinin synthase gene (ipt) was removed and replaced with a Cu-controllable promoter. Tobacco (Nicotiana tabacum L. cv tabacum) transformed with this Cu-inducible ipt gene (Cu-ipt) was morphologically identical to controls under noninductive conditions in almost all lines produced. However, three lines grew in an altered state, which is indicative of cytokinin overproduction and was confirmed by a full cytokinin analysis of one of these lines. The in vitro treatment of morphologically normal Cu-ipt transformants with Cu2+ resulted in delayed leaf senescence and an increase in cytokinin concentration in the one line analyzed. In vivo, inductive conditions resulted in a significant release of lateral buds from apical dominance. The morphological changes seen during these experiments may reflect the spatial aspect of control exerted by this gene expression system, namely expression from the root tissue only. These results confirmed that endogenous cytokinin concentrations in tobacco transformants can be temporally and spatially controlled by the induction of ipt gene expression through the Cu-controllable gene-expression system.

A system for tissue-specific copper-controllable gene expression in transgenic plants: nodule-specific antisense of aspartate aminotransferase-P2.

A vector system, based on copper controllable gene expression, has been developed to give control over place as well as time of expression of an introduced gene. This system consists of two elements: (1) the yeast ace1 gene encoding a metallo-regulatory transcription factor, ACE1, under control of either an organ-specific or a constitutive promoter; and (2) a gene of interest under control of a chimaeric promoter consisting of the 46 bp TATA fragment of the CaMV 35S RNA promoter linked to four repeats of the ACE1 binding site. The functioning of the system in an organ-specific manner was tested in nodulated Lotus corniculatus plants which consisted of non-transformed shoots plus transformed hairy root tissue 'wild-type tops/transgenic roots'. After addition of copper ions to the plant nutrient solution, beta-glucuronidase (GUS) expression was visualized either specifically in nodules or in both roots and nodules when the ace1 gene was placed under control of the nod45 promoter or the CaMV 35S RNA promoter, respectively. The nodule-specific system was used to express antisense constructs of aspartate aminotransferase-P2 in transgenic Lotus corniculatus plants. When expression was induced by the addition of copper ions to the plant nutrient solution aspartate aminotransferase-P2 activity declined dramatically, and a decrease of up to 90% was observed in nodule asparagine concentration.

Isolation, DNA sequence analysis, and mutagenesis of a proline dehydrogenase gene (putA) from Bradyrhizobium japonicum.

We report here the cloning and sequencing of the gene for proline dehydrogenase (putA) of Bradyrhizobium japonicum. An open reading frame coding for 1,016 amino acids was identified. The B. japonicum gene codes for a bifunctional protein with proline dehydrogenase and pyrroline-5-carboxylate (P5C) dehydrogenase activities, as it does in Escherichia coli and Salmonella typhimurium. Comparison of the sequences of these proteins with other proline and P5C dehydrogenase sequences identified proline dehydrogenase and P5C dehydrogenase catalytic domains. Within the proline dehydrogenation domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Saccharomyces cerevisiae put1, and Drosophila melanogaster slgA. Within the P5C dehydrogenase domain, several areas of high identity were observed between B. japonicum, E. coli, S. typhimurium, Bacillus subtilis ipa76d, and S. cerevisiae put2. A consensus catalytic site for semialdehyde dehydrogenase was observed in the P5C dehydrogenase domain. This suggests that the substrate for this domain may be the open-chain gamma-glutamylsemialdehyde, not its cyclized form, P5C. Unlike the gene isolated from E. coli, S. typhimurium, and K. pneumoniae, the B. japonicum putA gene does not appear to be part of an operon with the proline porter gene (putP). Additionally, the B. japonicum gene lacks the putative C-terminal regulatory domain present in the E. coli and S. typhimurium genes. The gene was disrupted by insertion of antibiotic resistance gene cassettes, which were then recombined into the bacterial chromosome. Symbiotically active mutant strains that were devoid of putA activity were isolated. With this proline dehydrogenase clone, we will test the hypothesis that putA in symbiotic nitrogen-fixing B. japonicum bacteroids is transcriptionally regulated by drought and other stresses.

Copper-controllable gene expression system for whole plants.

We describe a system for gene expression in plants based on the regulation mechanism of the yeast metallothionein (MT) gene. The system consists of two elements: (i) the yeast ace1 (activating copper-MT expression) gene encoding a transcription factor under control of the cauliflower mosaic virus (CaMV) 35S RNA promoter, and (ii) a gene of interest under control of a chimeric promoter consisting of the 90-base-pair domain A of the CaMV 35S RNA promoter linked to the ACE1 transcription factor-binding site. At elevated copper ion concentrations, the ACE1 protein changes conformation, binds to, and activates transcription from the chimeric promoter. To test the functioning of the system in plants, a construct containing the beta-glucuronidase (GUS) reporter gene under control of the chimeric promoter was prepared, and transgenic tobacco plants were produced. It was shown that GUS activity in the leaves of transgenic plants increased up to 50-fold, either after addition of 50 microM CuSO4 to the nutrient solution or after application of 0.5 microM CuSO4 to the plants in a foliar spray. This GUS expression was repressed after the removal of copper ions. The results show that the activity of the described chimeric promoter directly depends on copper ion concentration and that this system can be used in experiments that demand precise timing of expression.

[Mapping the genome locus containing genes for mouse tumor necrosis factor by means of hybridization end labelling with the use of synthetic oligonucleotides]. / Kartirovanie genomnogo lokusa, soderzhashchego geny faktorov nekroza opukholei myshi, metodom gibridizatsionnoi kontsevoi metki s ispol'zovaniem sinteticheskikh oligonukleotidov.

We have developed a technique for restriction nuclease sites mapping in genomic DNA cloned into phage lambda vectors. Synthetic oligonucleotides homologous to the vector sequences and adjacent to the cloning site were used as hybridisation probes for indirect end labelling procedure. In addition, a number of oligonucleotides homologous to the sequences of tumour necrosis factor genes were used for mapping from the internal sites. As a result, a map of 35 kb genomic region on the chromosome 17 inside major histocompatibility complex and surrounding mouse tumour necrosis factor genes was constructed.

The use of bacterial genes encoding herbicide tolerance in constructing transgenic plants.

The modes of action of some of the best-studied and widespread herbicides are briefly reviewed. Particular attention is given to those herbicide-inhibited processes that bacteria and plants have in common. We describe bacterial mutant genes of herbicide resistance, peculiarities of their introduction into plants, and success in the construction of transgenic resistant plants.

[Regions of human genome-containing analogs of oncogenes and retroviral genes. II. A new class of retrovirus-like elements]. / Uchastki genoma cheloveka, soderzhashchie analogi onkogenov i genov retrovirusov. II. Novyi klass retrovirusopodobnykh élementov.

Earlier we have found that recombinant phage lambda gp5 contains the sequences homologous to v-mos oncogene and retroviruses. After the nucleotide sequence determination we have found the region with homology to U5 part of retroviral LTR. Adjacent to this region are sequences complementary to 3'-end of tRNAMet. Numerous transcripts reacting with subcloned U5 probe from gp5 are present in polyadenylated RNA fraction from human cells. The humane genome contains several copies of these region, with two of them residing in gp5 locus. On the basis of these data we deduced the presence in the human genome of a new class of retroviral-like elements, existing probably as part of new human endogeneous retrovirus (HuEV).

Human genome: proto-oncogenes and proretroviruses.

A brief review of the studies undertaken at the Laboratory for Molecular Bases of Oncogenesis (Institute of Molecular Biology, Moscow) till middle of 1984 is presented. The human genome contains multiple dispersed nucleotide sequences related to the proto-oncogene mos and to proretroviral sequences in tight juxtaposition to each other. From sequencing appropriate cloned fragments of human DNA in phage and plasmid vectors it follows that one of these regions, NV-1, is a pseudogene of proto-mos with partial duplications and two Alu elements intervening its coding sequence, and the other, CL-1, seems to be also a mos-related gene with a deletion of the internal part of the structural gene. CL-1 is flanked by a proretroviral-like sequence including tRNAiMet binding site and U5 (part of the long terminal repeat). The proretroviral-like sequences are transcribed in 21-35S poly(A)+RNA abundant in normal and malignant human cells. Two hypotheses are proposed: endogenous retroviruses take part in amplification of at least some proto-oncogenes; proto-oncogenes are inactivated via insertion of movable genetic elements and conversion into pseudogenes. Potential oncogenicity of a normal human genome undergoes two controversial influences: it increases due to proto-oncogene amplification and decreases due to inactivation of some of them.

Human nucleotide sequences related to the transforming gene of a murine sarcoma virus: studies with cloned viral and cellular DNAs.

A recombinant plasmid, pI26, has been constructed by cloning into pBR322 a transforming gene of murine sarcoma virus (a Moloney strain, clone 124, MSV) synthesized by detergent-treated virions. From this plasmid a XbaI-HindIII fragment has been isolated which contains only mos-specific sequences. This mos-specific probe has been used for screening a human gene library cloned in bacteriophage lambda Charon 4A. Of these, 19 clones have been isolated containing mos-related sequences. By physical mapping and molecular hybridization it has been shown that these sequences are neighboured by DNA regions related to Moloney murine leukemia virus. Recombinant phages have also been found containing human inserts related to MLV, not to the mos gene. The possible existence of murine-like endogenous retroviruses in the normal human genome, including that of a sarcoma type, is discussed. By Northern blotting, expression of the cellular c-mos gene has been detected in mouse liver treated with a hepatocarcinogen. The general significance of the suggested model for evaluating the relationship between chemical carcinogenesis and oncogene expression is discussed.