Involvement of sigma(S) in starvation-induced transposition of Pseudomonas putida transposon Tn4652.

Transpositional activity of mobile elements can be induced by different environmental stresses. Here, we present evidence that transposition of Tn4652 is elevated in stationary-phase Pseudomonas putida and suppressed in an isogenic sigma(S)-defective strain. We demonstrate that transcription from the Tn4652 transposase promoter is controlled by the stationary-phase-specific sigma factor sigma(S). To our knowledge, this is the first example of direct stationary-phase-specific regulation of a mobile element transposase. Data presented in this report support the idea that activation of transposition under stressful conditions could be an inducible process.

Construction and molecular analysis of gene transfer systems derived from bovine immunodeficiency virus.

Because lentiviruses are able to infect nondividing cells, these viruses might be utilized in gene therapy applications where the target cell does not divide. However, it has been suggested that the introduction of primate lentivirus sequences, particularly those of human immunodeficiency virus, into human cells may pose a health risk for the patient. To avoid this concern, we have constructed gene transfer systems based on a nonprimate lentivirus, bovine immunodeficiency virus. A panel of vectors and packaging constructs was generated and analyzed in a transient expression system for virion production and maturation, vector expression and encapsidation, and envelope protein pseudotyping. Virion preparations were also analyzed for transduction efficiency in a panel of human and nonhuman primary cells and immortalized cell lines. The virion preparations transduced most of the target cell types, with efficiencies up to 90% and with titers of unconcentrated virus up to 5 x 10(5) infectious doses/ml. In addition, infection of nondividing human cells, including unstimulated hematopoietic stem cells and irradiated endothelial cells, was observed.

Gene transfer systems derived from Visna virus: analysis of virus production and infectivity.

Efficient transfer of therapeutic genes into nondividing human cells can be accomplished by inserting the genes into lentiviruses and infecting the cells with the modified viruses. The most developed lentivirus gene transfer systems are based on HIV-1, but because of the widespread HIV epidemic, the use of HIV-based vectors for gene therapy may be associated with a safety risk. In an attempt to find another lentivirus which can transduce human cells and might be safer than HIV-1, we generated gene transfer constructs based on the sheep lentivirus Visna. Molecular analysis of the constructs in a transient production system indicated that Visna produced as many mature virus particles as did HIV-1. Moreover, the virus particles incorporated a heterologous surface protein marker-gene-containing vector RNAs as efficiently as did HIV-1. However, the Visna virus transduced target cells poorly because of defects in reverse transcription and integration of the vector. Further modifications must be made to the Visna gene transfer system if the system is to be used in clinical gene therapy applications.

Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells.

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR(+) cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin(-/lo)Sca-1(+) and c-kit(+)Sca-1(+) subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR(+)c-kit(+)Sca-1(+) bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR(+) cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

Comparative analyses of intracellularly expressed antisense RNAs as inhibitors of human immunodeficiency virus type 1 replication.

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3' long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with the env antisense RNA, followed by the pol complementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and env antisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than the transdominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

Intracellular expression of RNA transcripts complementary to the human immunodeficiency virus type 1 gag gene inhibits viral replication in human CD4+ lymphocytes.

Intracellular expression of antisense transcripts was evaluated for its potential to interfere with human immunodeficiency virus type 1 (HIV-1) replication. Retroviral vectors encoding HIV-1 psi-gag complementary sequences downstream of a selectable gene (neo, puromycin gene, or Lyt2 gene) were stable and yielded high titers. Human CEMSS T cells were transduced with amphotropic retroviral vectors to express RNA complementary to the psi-gag sequence of HIV-1. Replication of laboratory-adapted HIV-1 strains was inhibited by more than 1 order of magnitude (log10) in these transduced cells even at high inoculation doses (4 x 10(4) 50% tissue culture infective doses). Antisense-mediated anti-HIV efficacy was further demonstrated by survival of CD4+ cells in these cultures relative to controls. The level of anti-HIV-1 activity of the psi-gag antisense sequence correlated with the length of the antisense transcript. Maximal anti-HIV efficacy was observed with complementary sequence more than 1,000 nucleotides long, whereas transcripts less than 400 nucleotides long failed to inhibit HIV-1 replication. Expression of psi-gag antisense RNA also reduced HIV-1 JR-CSF replication 10-fold in primary CD4+ lymphocytes. These results obtained with a T-cell line and primary peripheral blood lymphocytes indicate the potential of long antisense RNAs as an efficient anti-HIV-1 therapeutic agent for gene therapy.

Retroviral vectors designed for targeted expression of RNA polymerase III-driven transcripts: a comparative study.

Retroviral gene delivery systems for RNA polymerase II (RNA pol II)-based promoters have been developed and are widely used in gene transfer studies. In contrast, gene delivery systems with RNA pol III-based expression cassettes have not been studied comprehensively, although therapeutic applications (e.g., ribozymes, antisense, triplex RNA and RNA decoys) have been proposed. In this report, we describe retroviral vectors designed to optimize expression of short chimeric RNAs transcribed from a number of RNA pol III promoters. Our results show that all analysed RNA pol III expression cassettes (tRNA, U6, Ad VA1), regardless of orientation, do not transcribe efficiently when located between the retroviral long terminal repeats (LTRs). In contrast, high steady-state expression levels can be achieved by inserting the RNA pol III expression cassette into the U3 region of the LTR (double-copy design). Compared to human tRNA gene promoters (tRNA(Met), tRNA(Val)), the human small nuclear RNA U6 gene (U6) and the adenovirus virus-associated RNA 1 (Ad VA1) gene promoters yielded higher expression levels. The majority of the chimeric U6-derived transcripts were detected in the nuclear RNA fraction, and the VA1 and tRNA-driven transcripts were predominantly detected in the cytoplasmic compartments. This report is the first comparative study of RNA pol III-driven promoters expressing short chimeric transcripts leading to an optimized retroviral-vector design.

Translation of the rat LINE bicistronic RNAs in vitro involves ribosomal reinitiation instead of frameshifting.

The genomic structure of the rat LINE (L1Rn) DNA element contains two overlapping open reading frames (ORFs) and apparently has a potential to code for a DNA/RNA-binding protein (in ORF1) and a reverse transcriptase (in ORF2). We have characterized a 1,630-bp L1Rn cDNA clone encompassing the overlapping ORFs and a 600-bp genomic fragment derived from a full-length L1Rn member and containing the beginning of ORF1. These DNAs were used to restore in part the ORF1-ORF2 organization of L1Rn after being cloned into the pSP65 vector under the control of SP6 polymerase promoter. To test whether L1Rn ORF1 and ORF2 are expressed as a fusion protein, a series of capped RNAs with progressive truncations containing one or both ORFs were prepared and translated in the rabbit reticulocyte lysate. Our analysis indicates that the expression of a putative reverse transcriptase-encoded L1Rn ORF2 in vitro is regulated by reinitiation or internal initiation of translation but not by ribosomal frameshifting.

Consecutive DNA terminator sequencing by using enzymatically generated primers.

An improved strategy for the dideoxy chain termination sequencing of M13 recombinant DNA using enzymatically generated primers is presented. It involves synchronous extension of the universal primer with the Klenow fragment of DNA polymerase I at an average rate of 200 deoxynucleoside triphosphates per minute to the immediate downstream region of a preselected restriction enzyme site. Subsequent cleavage with the restriction enzyme generates the short primers with homogeneous 5' ends which can be used for further sequencing. The next restriction sites are selected in the newly sequenced regions of DNA by means of a microcomputer. By repeating this primer extension-cleavage-sequencing strategy sequences altogether about 6 kb long from several recombinant single-stranded M13 DNAs have been determined by using twenty restriction enzymes with different specificities.