James W. Gautsch

Improved technique utilizing nonfat dry milk for analysis of proteins and nucleic acids transferred to nitrocellulose

A simple, economical, and efficient procedure for analysis of proteins (Western blotting) and DNA (Southern blotting) transferred to nitrocellulose for reaction with antibodies or nucleic acid probes is described. The techniques utilize nonfat dry milk as a protein-nucleic acid source for blocking nonspecific reactions, as an incubation medium, and for subsequent washing to remove unreacted reagents. The incubation cocktail, termed BLOTTO (Bovine Lacto Transfer Technique Optimizer), is superior to bovine serum albumin or gelatin for preventing nonspecific absorption in Western blot analyses and does not require the use of detergents or chaotropic agents to effect efficient reduction of background. BLOTTO, at the proper dilution in NaClNa citrate, is just as efficient in Southern blot analyses as more complicated cocktails typically used in the latter technique. We also found that BLOTTO works well for blocking, incubating, and washing ELISA plate assays relative to the normal BSA carrier, at a considerable savings to the laboratory.

Differential expression of two distinct xenotropic viruses in NZB mice.

Abstract Two distinct xenotropic viruses have been isolated from New Zealand Black (NZB) mice. One of these viruses (NZB-X1) is spontaneously produced by NZB and NZB hybrid mice. The second xenotropic virus (NZB-X2) is induced by treatment of NZB fibroblasts with IdU. NZB-X1 is distinct in that it possesses a unique surface glycoprotein (gp70) similar to gp70 found free in the serum of all mouse strains tested, but only associated with a virion in NZB mice. NZB-X2 has a gp70 similar to that of xenotropic viruses isolated from mouse strains other than NZB, including xenotropic viruses of NIH Swiss, AKR, NZW, C57BL 6 BALB c , and wild mice, although NZB-X2 gp70 shows more structural relatedness to NIH Swiss and wild mouse xenotropic gp70 than to the others. By backcrossing NZB to NFS Swiss mice, two independently segregating loci have been described which control high and low xenotropic virus expression. Analyses of virus isolates from mice possessing these segregated loci showed that the high expression locus is linked to expression of the spontaneously released NZB virus, NZB-X1. The presence of the low expression locus corresponds with the production of the second xenotropic virus released after IdU treatment, NZB-X2. Using unique structural markers for these two xenotropic viruses, we have examined the phenomena of differential expression of endogenous virus and viral antigens within the NZB mouse. We find, as reported previously, that the predominant gp70 of serum is similar to NZB-X1. However, gp70s expressed on thymus and spleen carry the determinants of NZB-X2. The expression of these gp70s was not a peculiarity of NZB mice, since others tested including C57BL 6 , NZW, and 129 mouse strains showed the same tissue segregation. The differential expression of these xenotropic virus gp70s noncoordinate with virus production points to differential expression of endogenous envelope genes along different differentiation pathways.

Silent infection of murine embryonal carcinoma cells by moloney murine leukemia virus

Abstract Undifferentiated and differentiated cells from a teratocarcinoma of 129 mice do not exhibit spontaneous or bromodeoxyuridine-inducible virus or viral protein production. The differentiated cells can easily be productively infected while the undifferentiated embryonal carcinoma cells appear to be refractory. When the virus-exposed embryonal carcinoma cells are induced to differentiate and then treated with bromodeoxyuridine, infectious virus is readily recovered even after several months. Fifty percent of the single cell subclones are positive for inducible virus suggesting that infection is a common occurrence and that lack of virus expression is due to repression of the viral information.

B-MuX: a unique murine C-type virus containing the "env" gene of xenotropic viruses and the "gag" gene of the ecotropic virus.

Abstract A unique C-type virus, B-MuX, was isolated from nonproducer, BALB/c-transformed fibroblasts after IdUrd induction. B-MuX has the envelope gp70 properties of the endogenous BALB:virus-2 genome as determined by neutralization, interference, host-range characteristics and competition radioimmunoassays. Tryptic peptide analysis of gp70 from B-MuX revealed that B-MuX gp70 contains peptides in common to xenotropic virus gp70s and overall was very similar in structure to the xenotropic BALB: virus-2 gp70. In contrast, the gag gene products are serologically identical with the p15 and p12 of the endogenous, ecotropic virus. The p30 of B-MuX is structurally similar to that of the endogenous, ecotropic virus, and differs from the p30 of BALB/c xenotropic viruses as determined by tryptic peptide analysis. The results suggest that either B-MuX is a new type of endogenous, xenotropic virus or a recombinant between the endogenous xenotropic and ecotropic viruses. The unaltered xenotropic behavior of B-MuX demonstrates that the gag gene region does not contribute to this virological property.

A short-term quantitative XC assay for murine leukemia virus.

Abstract A syncytial-plaque (SXC) assay for murine leukemia virus is described which utilizes a single cycle of infection, thus allowing quantitation of virus-producing mouse cells in 3 to 4 days. Depending upon the Fv -1 genotype of infected mouse cells, the assay will reflect single or multiple “hitness” after infection by N- or B-tropic MuLV. The test is equally sensitive to the uv-XC plaque assay for tissue culture adapted MuLV, but is less sensitive to in vivo derived MuLV from an AKR/J mouse. Newly isolated AKR MuLV requires a longer latent period than the in vitro adapted Moloney strain of MuLV.

Steroid hormones increase the growth of MuLV in rat tumor XC cells.

Abstract Ecotropic MuLV, which do not normally grow efficiently in XC cells, were found to do so under the influence of specific steroid hormones. The adsorption of virus to the cells was not facilitated by the steroids. The increase in MuLV production by XC cells involves an increased viral progeny output per cell, thereby defining an intracellular restriction by rat cells on ecotropic MuLV growth. Cell lines other than the XC were not similarly stimulated. The steroid-induced increase in MuLV growth is accompanied by enhanced XC cell fusion.

Structural diversity among retroviral gene products: a molecular approach to the study of biological function through structural variability.

Publisher Summary The chapter presents the structural comparisons, the relationships between the various mammalian retroviruses, defines the extent to which the retroviruses represent a polymorphic family of genes. The peptide mapping techniques and the data obtained by its use concerning the viruses of the family Retroviridae and to integrate this information with that obtained through different methods by others. The use of refined methods in peptide mapping technology to study the gene products of type B, C, and D retroviruses, especially MuLV, has enabled defining of markers on the viral gene products of most extant retrovirus isolates. This information, as a whole, has provided insight into the range of variability that exists among the mammalian retroviruses and allows the identification of virtually any unknown virus, its host range, Fv-1 type, and endogenous origin. Structural functional correlates have been noted in the chapter. The instances of intergenic and intragenic recombination have been identified using the structural markers on MuLV gp70, p30, and p15, and this evidence has presented the suggestions.

Lack of retrovirus gene expression in teratocarcinoma stem cells is limited to the nucleus

Experiments are described showing that the block to retrovirus replication by undifferentiated embryonal carcinoma cells can be overcome by fusion with differentiated cell karyoplasts but not cytoplasts. This supports previous notions that the expression of input proviral genes in teratocarcinoma stem cells is under different controls than in differentiated cells and indicates that the exercising of this control is limited to the nucleus of the embryonal carcinoma cell.