Biomedical subjects
Publications and source records attributed to W P Rowe.
A combination of somatic cell hybridization and standard mendelian breeding techniques was used to map the AKR ecotropic virus inducibility locus Akv-2 to the centromeric end of chromosome 16. This assignment of Akv-2 further emphasizes the endogenous ecotropic retroviruses are inserted at multiple sites in mouse chromosomes.
A single mendelian gene was identified for induction of the endogenous xenotropic murine leukemia virus in five mouse strains (C57BL/10, C57L, C57BR, AKR, and BALB/c). This locus, designated Bxv-1, mapped to the same site on chromosome 1 in all strains: Id-1-Pep-3-[Bxv-1-Lp]. Thus, inducibility loci for xenotropic virus are more limited in number and chromosomal distribution than ecotropic inducibility loci. Virus expression in mice with Bxv-1 was induced by treatment of fibroblasts with 5-iododeoxyuridine or by exposure of spleen cells to a B cell mitogen, bacterial lipopolysaccharide. An analysis of the hamster X mouse somatic cell hybrids indicated that chromosome 1, alone, was sufficient for virus induction.
Recombinant mink cell focus-inducing (MCF) murine leukemic viruses, as well as ecotropic and xenotropic viruses, were tested for ability to accelerate or cause development of lymphoma in AKR and other strains of mice. Of the three classes of virus isolated from AKR, only the MCF viruses were able to accelerate development of AKR lymphoma. This fully supports the idea that the MCF viruses are the proximal cause of spontaneous AKR lymphoma. MCF lymphomagenicity was strain specific, however, in that AKR MCF viruses did not induce lymphomas in many murine strains; they were moderately lymphomagenic in C3H/Bi mice and in National Institutes of Health Swiss partially congenic for Akv-1 or Akv-2. In contrast, MCF viruses from nonthymic hematopoietic neoplasms of C3H/Fg, BALB/c, or mice partially congenic for ecotropic virus loci (Akv-1, Akv-2, Fgv-1, C58v-1, and C58v-2) were not able to accelerate or cause lymphomia in AKR or any other mouse strain tested, including some of the strains of origin. MCF lymphomagenicity correlated with thymic origin in the virus and with ability to replicate in the thymus.
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A specific probe for detecting ecotropic murine leukemia virus sequences was constructed by cloning a 500-base-pair DNA segment, corresponding to a portion of the env region of the AKR ecotropic virus, in a pBR322/Escherichia coli K-12 host/vector system. This probe was used to screen the cellular DNAs of six inbred strains of mice for the presence of ecotropic retroviral DNA sequences by the Southern blot hybridization procedure. Three copies of ecotropic viral DNA were detected in AKR/N (a high-ecotropic virus strain) and two were found in BALB/c (a low-ecotropic virus strain) DNAs. As expected, no sequences reactive with this probe were found in NFS mouse DNA (a virus-negative strain). However, cellular DNA sequences that reacted strongly with the ecotropic-specific DNA probe were detected in certain NZB, C57L, and 129 mice (all virus-negative strains). In contrast to the reactive sequences in AKR and BALB/c, the reactions were chiefly associated with EcoRI segments that were subgenomic in size.
Congenic mouse strains NIH,Akv-1 and NIH,Akv-2 carry the two high ecotropic virus-inducing loci of the AKR mouse on the NIH Swiss genetic background. Progeny tests of animals in three separate congenic families show that both Avk-1 and Akv-2 are stably transmitted as classical mendelian loci in these mice. However, during the process of inbreeding, additional chromosomal viral loci were detected in several NIH.Akv-1 sublines. These loci appeared only in the progeny of virus-positive females. They segregate with mendelian ratios, are unlinked to markers on chromsome 7 near Akv-1, and are phenotypically expressed as high-virus-inducing loci. The generation of new loci for viurs induction, no doubt resulting from the rare germ-line reintegration of the endogenous ectropic provirus, represents a unique form of gene duplication and rearrangement.
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By using seven different restriction endonucleases, the cleavage patterns of the unintegrated provioral DNA from an ecotropic murine leukemia virus isolated from Mus musculus molossinus were found to be identical to those of AKR virus. An AKR [3H]DNA probe can be completely saturated with M. musculus molossinus and M. musculus castaneus DNAs, although the arrangement of viral sequences in M. musculus molossinus DNA differed from that of AKR virus. These studies indicate that an AKR-type ecotropic virus is present in some wild Asiatic mice.
Inoculation of suckling hamsters with 2 x 10(8) live cells of Escherichia coli K12 strain chi1776, carrying the complete genome of polyoma virus in a recombinant plasmid, failed to induce tumors in any of 32 recipients. Also, lambda phage DNA and particles with a monomeric insert of polyoma DNA did not induce tumors. Purified recombinant plasmid DNA, as well as phage particles and DNA containing a head-to-tail dimer of polyoma DNA, showed a low degree of oncogenicity, comparable to that of polyoma DNA prepared from mouse cells. These findings support the previous conclusions, based on infectivity assays in mice, that propagation of polyoma virus DNA as a component of recombinant DNA molecules in E. coli K12 reduces its biologic activity many orders of magnitude relative to the virus itself.
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By means of an approach that combined the techniques of somatic cell genetics and Mendelian breeding studies, the inducibility locus, designated Cv, for ecotropic murine leukemia virus in BALB/c mice, was mapped to chromosome 5, 23 units from the locus for phosphoglucomutase-1, with gene order Cv-Pgm-1-Gus. This low-efficiency inducibility locus is therefore not allelic with the chromosome 7 loci previously described for two other mouse strains with high virus inducibility. These studies provide further evidence that endogenous ecotropic viruses represent viral genomes inserted at different chromosomal sites in the various mouse strains.
A series of recombinant plasmids containing polyoma virus (PY) DNA were constructed, and their biological activity was evaluated in mice and in cultured mouse cells. While all of the recombinants studied contain the complete, potentially infectious viral DNA, in no case was the intact recombinant PY-plasmid DNA, or live Escherichia coli containing the recombinant plasmids, capable of inducing PY infection of mice, either by feeding or by parenteral injection.
The biological activity of recombinant phage and recombinant phage DNA containing monomeric or dimeric polyoma DNA inserts was examined in mice and cultured mouse cells. Recombinant preparations containing a single copy of viral DNA were invariably noninfectious; molecules containing a dimeric polyoma DNA insert were at least seven orders of magnitude less infectious than polyoma virions after parenteral inoculation. No infection was detected with any recombinant preparation after oral administration.
Distinct type-specific antigens were detected on cells infected with cloned mink cell focus-inducing (MCF) murine leukemia viruses by means of cell surface immunofluorescence absorption assays with rabbit antisera raised against naturally-occurring AKR MCF viruses. The MCF type-specific antibodies were present in high titer and not absorbable by cells infected with ecotropic, xenotropic, or wild mouse amphotropic murine leukemia viruses, or combinations of ecotropic and xenotropic viruses. Three MCF subtype-specific reactions were identified. One subspecificity (operationally designated MCFA-1) defined antigenic determinant(s) distributed among MCF viruses in general. Another (MCFA-2) specified determinant(s) induced by all naturally occurring MCF isolates not of Friend or Moloney origin. A third subspecificity (MCFA-3) was induced by some MCF isolates, and not by others; the presence of this antigen did not correlate with the source of any presently known biological property of the viruses. In addition, type-specific antigenic determinants of ecotropic and xenotropic murine leukemia viruses were expressed on MCF virus-infected cells. The serological profile of MCF viruses thus supports the contention that they are env gene recombinants between ecotropic and xenotropic murine leukemia viruses. However, new, distinct MCF-specific determinants are also generated, and these could be useful markers in studying MCF viruses.
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The tumorigenicity of DNA from polyoma virus after cleavage with a variety of restriction enzymes was evaluated in suckling hamsters. Cleavage with enzymes that interrupt the region of the genome coding for the large tumor (T) antigen of polyoma virus markedly enhanced the tumorigenicity above that observed with DNA I of the virus. Cell lines established in vitro from tumors induced by polyoma virions, polyoma virus DNA I, or polyoma virus DNA that had been cleaved with restriction endonucleases in the early region all contain the polyoma virus middle and small T antigens but not the large T antigen of polyoma virus is not required for maintenance of the transformed state and probably not for initiation of tumorigenesis by viral DNA.
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.