Identification of DNA fragments carrying ecotropic proviruses of AKR mice.

Abstract

The proviruses of the N-tropic, ecotropic virus (AKV) of AKR mice (Akv-1, Akv-2) have been studied by the Southern gel-filter transfer technique. These proviruses can be detected by cleavage of cell DNA by BamHI endonuclease, which-yields characteristic subgenomic DNA fragments upon cleavage of this type of provirus. Proviruses integrated into different sites in the mouse genome can be resolved with EcoRI endonuclease, which does not cleave the AKV proviruses. Use of congenic and backcrossed mice and a radioactive DNA probe enriched for AKV sequences has allowed identification of the EcoRI fragments carrying the proviruses of the genetically defined Akv-1 and Akv-2 loci. Novel proviruses introduced by superinfection of cultured AKR cells with AKV and present in leukemic cells from AKR mice have also been identified. Comparison of substrains of AKR mice indicates some heterogeneity in their spectra of proviruses.The AKR strain of mouse is characterized by a life-long expression of an ecotropic N-tropic murine leukemia virus, AKV, and a 70-95% incidence of leukemia (1, 2). Both of these characteristics are the result of dominant alleles present at two loci, Akv-1 and Akv-2 (3). Congenic mice have been constructed that possess either the Akv-1 or Akv-2 allele in the genetic background of the NIH/Swiss strain of mouse (4), a strain which otherwise lacks such alleles. Hybridization of DNA from such congenic mice to an AKV cDNA probe demonstrated that the Akv-1 allele segregates with and presumably represents a provirus for AKV. The allele at Akv-2 is associated with a second, apparently identical (5), provirus.The proviruses present at Akv-1 and Akv-2 are not necessarily expressed. Embryonic AKR mice and many cell lines derived from AKR embryos do not produce virus. In vitro, expression of virus occurs at a low frequency spontaneously and at a much higher frequency upon induction with BrdUrd (6). Because AKR cells are sensitive to exogenous infection with AKV, such transient induction of virus expression leads to horizontal infection of the entire culture and subsequent continuous virus production. In vivo, expression of virus occurs from some cells in all postnatal AKR mice. The in vivo expression of virus is probably also the result of transient induction of virus by a few cells followed by horizontal infection of other cells in the animal.The number of AKV proviruses present in virus-negative, virus-producing, and leukemic AKR cells has been estimated by liquid hybridization (7,8). This technique allows relatively accurate quantitation of the amount of AKV sequences present in a given DNA sample. It provides no information, however, about the organization of these sequences. Recently, the Southern gel-filter transfer technique (9) has been used successfully to analyze single integrated murine leukemia virus (MuLV) proviruses in rodent cells (10). We apply this technique here to identify the Akv-1 and Akv-2 genetic loci with specific DNA fragments produced by restriction endonucleases, to identify specific ...

Generation and analysis of clonal IgM- and IgG-producing human B cell lines expressing an anti-DNA-associated idiotype.

“…Cell lines were assayed for clonality by Southern blot analysis using specific human JK and JH probes (Table I). High molecular weight DNAs from the EBV lines were prepared essentially as described by Steffen et al (6). Briefly, 107 cells were suspended in 5 ml of 100 mM NaCl 10 mM Tris-HCl (pH 7.5) and 1 mM EDTA.…”

Proviruses are adjacent to c-myc in some murine leukemia virus-induced lymphomas.

“…[32P]DNA as described (9). DNA of plasmid pv-myc3 was 32P-labeled (to 4 x 108 dpm/,ug) by nick-translation (10) and was used at 2 ,uCi/ml (1 Ci = 37 GBq) to detect the c-myc gene.…”

Regulation of transcription of the adenovirus EII promoter by EIa gene products: absence of sequence specificity.

“…High-molecular-weight cellular DNA was isolated as described by Steffen et al (32). DNA was digested with BamHI, and 10 ,ug was separated on a 1.0% agarose gel.…”