Vectors permitting visual monitoring of simple transposition events

The construction and use of two novel transposon(Tn)-delivery vectors is described. These vectors carry Inc.W or Inc.N broad-host-range transfer functions cloned next to the narrow-host-range replicon of pBR329. The host specificities of pSLX10 and pSLX23 both complement and extend the host specificities of existing Tn delivery vectors. Plasmids pSLX10 and pSLX23 were shown to transfer at high frequency in intergeneric matings. The lux genes which are present on each vector permit the visual monitoring of transconjugants which have retained a Tn element, but are devoid of plasmid molecules. pSLX10 and pLSX23 were efficiently used to generate a range of auxotrophic mutants in various strains of Pseudomonas as well as to clone genes from Serratia liquefaciens. These vectors may have general applicability to identify and clone genes in a wide range of Gram-negative bacteria.     

Insulin-regulated glucose uptake in rat adipocytes is mediated by two transporter isoforms present in at least two vesicle populations

We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.     

Cell-free transfer of sterols from dictyosome-like structures to plasma membrane vesicles of guinea pig testes

Summary Transfer of radiolabeled lipids from dictyosome-like structures (DLS) from testis tubules of the guinea pig as donor to unlabeled plasma membrane from testis tubules immobilized on nitrocellulose as acceptor was studied in a completely cell-free system. As a general label for lipids of the donor DLS, isolated testis tubules were incubated with [¹⁴C]acetate. Time- and temperature-dependent transfer of [¹⁴C]acetate labeled constituents was observed in the cellfree system. However, despite the fact that phospholipids and other constituents were highly labeled in the donor fraction, primarily radioactive sterols were transferred to the plasma membrane acceptor vesicles. Transfer at 37°C represented 0.4 to 0.7% of the total radiolabeled cholesterol at 37°C but little or no transfer occurred at 4°C. The sterols transferred exhibited Chromatographic mobilities corresponding to those of cholesterol and lanosterol. Similar results were obtained with [¹⁴C]mevalonic acid. In subsequent experiments, cholesterol transfer from DLS to plasma membrane was demonstrated by incubation of DLS with [³H]squalene which was converted into sterol or with [¹⁴C]cholesterol. Transfer of sterols required ATP, but not cytosol, and was both time- and temperature-dependent. DLS were more effective than either endoplasmic reticulum or plasma membrane as the donor fraction. The results from the cell-free analysis suggest a possible functional role of the DLS in sterol biogenesis and transfer to the plasma membrane during spermatid development.Abbreviations DLS dictyosome-like structure(s) - PBS phosphatebuffered saline - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - BSA bovine serum albumin     

A recombinant adenovirus expressing an Epstein-Barr virus (EBV) target antigen can selectively reactivate rare components of EBV cytotoxic T-lymphocyte memory in vitro.

While the bulk of a virus-induced cytotoxic T-lymphocyte (CTL) response may focus on a few immunodominant viral antigens, in certain tumor virus systems the detectability of clones recognizing other, subdominant antigens can assume particular importance. By using the human CTL response to Epstein-Barr virus (EBV) as a model system, here we show that even rare components of virus-specific memory can be selectively reactivated in vitro when the relevant target antigen is expressed in autologous stimulator cells from a recombinant adenovirus (RAd) vector. We generated a replication-deficient adenovirus, RAd-E3C, which in skin fibroblast cultures expressed the EBV nuclear antigen EBNA3C at a 10- to 100-fold-higher level than that naturally present in EBV-transformed lymphoblastoid cell lines (LCLs). Initial experiments with a donor whose polyclonal CTL response to LCL stimulation contained a strong EBNA3C-specific component showed that these CTLs could be efficiently reactivated by in vitro stimulation either with RAd-E3C-infected fibroblasts or with RAd-E3C-infected peripheral blood mononuclear cells. Then we studied donors whose responses to LCL stimulation contained little if any detectable EBNA3C reactivity but were dominated by clones recognizing other EBV target antigens; in vitro stimulation with RAd-E3C-infected peripheral blood mononuclear cells selectively reactivated EBNA3C-specific CTL clones from these individuals, with the epitope specificities of responses subsequently identified at the peptide level. This RAd-based approach could be applied more generally to screen for human CTL responses against any candidate target antigen expressed by tumor cells.     

A Protein Kinase-Dependent Block to Reinitiation of DNA Replication in G2 Phase in Mammalian Cells

Eukaryotic cells normally replicate their DNA only once between mitoses. Unlike G1 nuclei, intact G2 nuclei do not replicate during incubation inXenopusegg extract. However, artificial permeabilization of the nuclear membrane of G2 nuclei allows induction of new initiations byXenopusegg extract. This is consistent with the action of a replication licensing factor which is believed to enter the nucleus when the nuclear membrane breaks down at mitosis. Here, we show that G2 nuclei will initiate a new round of replication in the absence of nuclear membrane permeabilization, if they are preexposed to protein kinase inhibitorsin vivo.Competence to rereplicate is generated within 30 min of drug treatment, well before the scheduled onset of mitosis. This demonstrates that a protein kinase-dependent mechanism is continually active in G2 phase to actively prevent regeneration of replication capacity in mammalian cells. Kinase inhibition in G2 cells causes nuclear accumulation of replication protein A. Rereplication of kinase-inhibited G2 nuclei also depends on factors supplied byXenopusegg extract, which are distinct from those required for replication licensing.