Extrachromosomal copies of the 1.6-kilobase transposable element Tcl are present at the level of between 0.1 and 1.0 copy per cell in Caenorhabditis elegans strain Bergerac. Extrachromosomal elements were detected and studied using Southern hybridizations employing a Tcl-specific probe. The amount of extrachromosomal Tcl DNA was roughly constant during development in Bergerac, which has approximately 300 integrated chromosomal copies of Tcl in its haploid genome. Extrachromosomal Tcl DNA was not detected in strain Bristol, which has 30 chromosomal copies of TcW. Three forms of extrachromosomal DNA were detected. The predominant form was a 1.6-kilobase linear molecule with ends corresponding to the ends of an integrated Tcl element. The other two forms were, respectively, relaxed and supercoiled circular copies of the element. Structural assignments were based on electrophoretic mobility, the results of sedimentation velocity and equilibrium density gradient experiments, and on the sizes of the products produced by treatment of purified extrachromosomal DNA with restriction endonucleases. The suggestion is made that these extrachromosomal transposable elements are the products of excision events known to be occurring at high frequency in somatic cells in Bergerac. (strain N2) were from the laboratory of D. Hirsh. Culture conditions, including the preparation of synchronous populations, have been described (5, 6).DNA Isolation. The method of DNA isolation from nematodes was as described by Emmons and Yesner (5).Restriction Endonuclease Digestion, Gel Electrophoresis, and Southern Hybridization. Enzymes were obtained from New England Biolabs and were used following the supplier's specifications. Agarose (type II, medium electroendosmosis) was from Sigma. Electrophoresis was carried out in Tris borate buffer (89 mM Tris base/89 mM boric acid/2.5 mM Na2EDTA, pH 8.3). Transfer to nitrocellulose (Schleicher & Schuell) was according to Southern (7). The immobilized DNA was hybridized to a 32P-labeled DNA probe in a solution containing 50% formamide and dextran sulfate (8). Details of the hybridization procedure were as follows. The nitrocellulose filter was prehybridized (10 ml/100 cm2) in 50% (vol/vol) formamide (MCB, Cincinnati, OH)/3x SET buffer (20x SET = 3 M NaCl/0.02 M EDTA/1 M Tris buffer, pH 7.9)/0.1 M sodium phosphate buffer (pH 7.0)/5x BFP solution (lx BFP = 0.02% bovine serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone) containing denatured sonicated Escherichia coli DNA at 100 ,g/ml. Prehybridization was at 37°C for 2-4 hr. Hybridization was carried out in a similar solution, except that BFP solution was lx and sodium dodecyl sulfate (to 0.3%) and sodium dextran sulfate 500 [to 10% (wt/vol)] were added. Hybridization was at 37°C overnight. The hybridized filter was washed at 37°C with two or three changes of 2x SET buffer/0.1 M phosphate buffer, pH 7.0/0.2% sodium dodecyl sulfate, with 30 min between changes. Finally, the filter was rinsed in 0.2x SET buffer, dried, and exposed for 24-48 hr to ...

Eleven chromosomal products of somatic excision of Tc1 transposable elements have been cloned and sequenced. The cloning method did not involve genetic reversion; therefore the products analyzed should be representative. Six empty religated target sites were from excision of one Tc1 element inserted near actin genes on linkage group V; five were from a second Tc1 element inserted elsewhere on the same linkage group. All six products from the first element were identical in sequence to an empty target site from a second strain, indicating excision had been precise. Two of the products from the second element were also precise, whereas the other three contained four extra nucleotides at the point of excision, indicating an imprecise excision. The four nucleotides are the same in all cases and could represent two terminal nucleotides of the transposon plus a two-nucleotide target site duplication. The difference in the ratio of precise to imprecise excision at the two insertion sites suggests a possible chromosomal position effect on the pathway of Tc1 somatic excision.

No abstract

In an initial attempt to test the ability of replication-defective retroviruses to immunize against immunologically related pathogenic viruses, we have worked with the erythroleukemogenic Friend retrovirus complex (FV), which consists of a replication-competent helper component, Friend murine leukemia virus (FMuLV), and a related defective pathogenic component, spleen focus-forming virus (SFFV). An 81-base-pair deletion was introduced into the plSE-encoding region of the env gene of an otherwise replication-competent molecular clone of the FMuLV provirus. After transfection of this clone into cells that package the viral RNA in MuLV coats, infectious virus was released into the culture medium. Mouse fibroblasts infected with this virus, here called AFMuLV, expressed the truncated viral env gene products in their cytoplasm but not on cell surfaces, and culture fluids from these cells did not transmit the infection to fresh mouse fibroblasts. In preliminary experiments, immunization of mice of H-2-congenic BALB/c strains with AFMuLV conferred levels of inmnunity to FV dise ranging from weak to relatively strong.Immunized mice developed anti-FV IgM and IgG antibodies and cytotoxic T cells. Mice observed for 15 weeks after the first of two immunizations showed no detectable pathology, but AFMuLV DNA was detectable in livers of some immunized mice for at least 3-6 weeks. These results suggest that our approach to development ofretrovirus vaccines may be a useful one.

No abstract