Sequence organization of feline leukemis virus DNA in infected cells

A restriction site map has been deduced of unintegrated and integrated FeLV viral DNA found in human RD cells after experimental infection with the Gardner-Arnstein strain of FeLV. Restriction fragments were ordered by single and double enzyme digests followed by Southern transfer (1) and hybridization with 32P-labeled viral cDNA probes. The restriction map was oriented with respect to the 5' and 3' ends of viral RNA by using a 3' specific hybridization probe. The major form of unintegrated viral DNA found was a 8.7 kb linear DNA molecule bearing a 450 bp direct long terminal redundancy (LTR) derived from both 5' and 3' viral RNA sequences. Minor, circular forms, 8.7 kb and 8.2 kb in length were also detected, the larger one probably containing two adjacent copies of the LTR and the smaller one containing one copy of the LTR. Integrated copies of FeLV are colinear with the unintegrated linear form and contain the KpnI and SmaI sites found in each LTR

The baboon endogenous virus genome. II. Provirus sequence variations in baboon cell DNA

Restriction analysis of the approximately 100 integrated baboon endogenous virus (BaEV) proviruses in baboon cells and tissues has revealed two major sequence variations, both in the gag gene region of the genome. One, a 150 nucleotide pair insert, is present in a small proportion of the proviral DNAs of some baboons, but is present in the majority of the proviral DNAs of other baboons. The second, a Bam HI recognition sequence located 2.25 kb from the proviral 5' end, is missing or modified in approximately one-half of the integrated genomes. We consider the possibility that accumulation of proviruses not containing the 0.15 kb insert is correlated with viral activation and expression since it is this form that is a replication intermediate in freshly infected permissive cells. It is evident from these initial studies that the organization of the multiple BaEV proviruses in baboon DNA has undergone modification during evolution

Baboon endogenous virus genome. I. Restriction enzyme map of the unintegrated DNA genome of a primate retrovirus

A detailed restriction map was deduced for the genome of an endogenous retrovirus of a higher primate, that of baboon. The cleavage sites for 12 restriction enzymes were mapped. The unintegrated linear viral DNA intermediate that is produced by infection of permissive cells with baboon endogenous virus was isolated. Hybridization with a strong-stop complementary DNA probe demonstrated presence of a terminal repetition in the linear viral DNA. The positions of restriction sites for two particular enzymes, SmaI and XhoI, near each end were consistent with this result and indicated that the length of the repetition is 0.55 +/- 0.01 kilobase. The linear viral DNA had a unique restriction map indicating that it is not a set of random circular permutations of the RNA genome. From hybridization with a 3'-specific probe, the DNA restriction map was aligned relative to the 5'-to-3' orientation of the viral RNA. We observed a minor heterogeneity in a BamHI recognition site 1.95 kilobases from the right end of the linear map

Baboon endogenous virus genome: Molecular cloning and structural characterization of nondefective viral genomes from DNA of a baboon cell strain

Several heterogeneities in the baboon endogenous virus (BaEV) genomes that are present in the DNA of normal baboon tissues and the baboon cell strain BEF-3 have been described previously. To study these genomes, we cloned BaEV proviruses from BEF-3 cellular DNA into the vector Charon 4A. Of the four full-length clones isolated, one was nondefective as determined by transfection. The sequence of a portion of this clone was found to code for amino acids 61-91 in the p30 region of the gag gene. This identification allowed us to align the restriction map with the BaEV genetic map. One heterogeneity, a BamHI site 2.4 kilobases (kb) from the proviral 5' end, was located close to the gag-pol junction; another, a BamHI site 1.4 kb from the 5' end of the genome, corresponded to the gag p30 coding sequence for amino acids 32-34; and a third, a Xho I site, was near the 3' end of the pol gene. To select the nondefective BaEV genomes from BEF-3 cells, we infected permissive cells with virus produced by BEF-3 cells and also transfected BEF-3 cellular DNA into permissive cells. The BaEV genomes in the permissive recipient cultures were then analyzed by restriction enzyme analysis. These nondefective genomes were found to be heterogeneous with respect to the gag-pol BamHI site and the Xho I site, but all were found to contain the BamHI site 1.4 kb from the 5' end of the genome

Identification of a BALB/c H-2L^d gene by DNA-mediated gene transfer

Gene transfer and immunoselection were used in the identification of a BALB/c genomic clone containing an H-2L^d gene (clone 27.5). Transformation of thymidine kinase-negative C3H mouse L cells with the cloned 27.5 DNA together with the herpes simplex virus tk gene produced transformants expressing L^d molecules detected by radioimmune assay with monoclonal hybridoma antibodies to Ld antigens. The foreign L^d gene products expressed by cloned mouse L cell transformants were shown to be virtually indistinguishable from BALB/c spleen L^d molecules by two-dimensional electrophoretic analysis of H-2L^d immunoprecipitates. These results indicate that the genomic clone 27.5 contains a functional BALB/c H-2L^d gene and demonstrate the usefulness of this approach for identifying the gene products encoded by cloned genes which are members of a multigene family. Furthermore, the ability to place cell-surface recognition molecules on the surfaces of foreign cells provides a powerful opportunity for functional analyses of these molecules

Germline transmission of exogenous genes in the chicken

Difficulties associated with in vitro manipulation and culture of the early chicken embryo have restricted generation of transgenic chickens to approaches that use replication-competent retroviruses. The need to produce transgenic chickens in the absence of replicating virus prompted development of a new method of gene transfer into the chicken. Microinjection of the replication-defective reticuloendotheliosis virus (REV) vector ME111 beneath unincubated chicken embryo blastoderms results in infection of germline stem cells. This vector contains genetic information exogenous to the chicken genome, including both the herpes simplex virus type 1 thymidine kinase gene and the Tn5 neomycin phosphotransferase gene. About 8 percent of male birds hatched from injected embryos contained vector DNA in their semen. All four positive males tested passed vector sequences onto their progeny. Analysis of G1 offspring showed that gonads of G0 male birds were mosaic with respect to insertion of vector provirus. Thus, primordial germ cells present in the unincubated chicken embryo blastoderm are susceptible to infection by defective REV vectors