Genetically controlled variation of "acid" beta-galactosidase detected in Rattus norvegicus by isoelectric focusing.

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of beta-galactosidases derived from different organs. The beta-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of beta-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same beta-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" beta-galactosidase.

Detection and sequence analysis of a nickel-induced mutation in a retroviral model system

We have developed a novel way to assess the mutagenicity of environmentally important metal carcinogens, such as nickel, by creating a positive selection system based upon the conditional expression of a retroviral transforming gene. The target gene is the v-mos gene in MuSVts110, a murine retrovirus possessing a growth temperature dependent defect in expression of the transforming gene due to viral RNA splicing. In normal rat kidney cells infected with MuSVts110 (6m2 cells), splicing of the MuSVts110 RNA to form the mRNA from which the transforming protein, p85$\sp{\rm gag-mos}$, is translated is growth-temperature dependent, occurring at 33 C and below but not at 39 C and above. This splicing "defect" is mediated by cis-acting viral sequences. Nickel chloride treatment of 6m2 cells followed by growth at 39 C, allowed the selection of "revertant" cells which constitutively express p85$\sp{\rm gag-mos}$ due to stable changes in the viral RNA splicing phenotype, suggesting that nickel, a carcinogen whose mutagenicity has not been well established, could induce mutations in mammalian genes. We also show by direct sequencing of PCR-amplified integrated MuSVts110 DNA from a 6m2 nickel-revertant cell line that the nickel-induced mutation affecting the splicing phenotype is a cis-acting 70-base duplication of a region of the viral DNA surrounding the 3$\sp\prime$ splice site. These findings provide the first example of the molecular basis for a nickel-induced DNA lesion and establish the mutagenicity of this potent carcinogen. ^

Identification of cadmium-induced mutations in a mammalian cell line

Epidemiological studies have shown cadmium to induce cancer in humans, while experimental studies have proven this metal to be a potent tumor inducer in animals. However, cadmium appears nonmutagenic in most prokaryotic and eukaryotic mutagenesis assays. In this study, we present the identification of mutations in normal rat kidney cells infected with the mutant MuSVts110 retrovirus (6m2 cells) as a result of treatment with cadmium chloride. The detection of these mutations was facilitated by the use of a novel mutagenesis assay established in this laboratory. The 6m2 reversion assay is a positive selection system based on the conditional expression of the MuSVts110 v-mos gene. In MuSVts110 the gag and mos genes are fused out of frame, thus the translation of the v-mos sequence requires a frameshift in the genomic RNA. In 6m2 cells this frameshift is accomplished by the temperature-dependent splicing of the primary MuSVts110 transcript. Splicing of MuSVts110, which is mediated by cis-acting sequences, occurs when 6m2 cells are grown at 33$\sp\circ$C and below, but not at 39$\sp\circ$C. Therefore, 6m2 cells appear transformed at low growth temperatures, but take on a morphologically normal appearance when grown at high temperatures. The treatment of 6m2 cells with cadmium chloride resulted in the outgrowth of a number of cells that reverted to the transformed state at high growth temperatures. Analysis of the viral proteins expressed in these cadmium-induced 6m2 revertants suggested that they contained mutations in their MuSVts110 DNA. Sequencing of the viral DNA from three revertants that constitutively expressed the P85$\sp{gag{-}mos}$ transforming protein revealed five different mutations. The Cd-B2 revertant contained three of those mutations: an A-to-G transition 48 bases downstream of the MuSVts110 3$\sp\prime$ splice site, plus a G-to-T and an A-to-T transversion 84 and 100 bases downstream of the 5$\sp\prime$ splice site, respectively. The Cd-15-5 revertant also contained a point mutation, a T-to-C transition 46 bases downstream of the 5$\sp\prime$ splice site, while Cd-10-5 contained a three base deletion of MuSVts110 11 bases upstream of the 3$\sp\prime$ splice site. A fourth revertant, Cd-10, expressed a P100$\sp{gag{-}mos}$ transforming protein, and was found to have a two base deletion. This deletion accomplished the frameshift necessary for v-mos expression, but did not alter MuSVts110 RNA splicing and the expression of p85$\sp{gag{-}mos}.$ Lastly, sequencing of the MuSVts110 DNA from three spontaneous revertants revealed the same G to T transversion in each one. This was the same mutation that was found in the Cd-B2 revertant. These findings provide the first example of mutations resulting from exposure to cadmium and suggest, by the difference in each mutation, the complexity of the mechanism utilized by cadmium to induce DNA damage. ^

A TEMPERATURE-SENSITIVE DEFECT IN THE EXPRESSION OF A SARCOMA VIRUS TRANSFORMING GENE

The viral proteins synthesized by a Moloney murine sarcoma virus (Mo-MuSV) with a temperature-sensitive mutation in a function required for the maintenance of the transformed state (ts110) were examined. Normal rat kidney cells (NRK) were infected with the ts110 virus and a non-virus-producing cell clone, termed 6m2, was isolated. This cell clone had a malignant phenotype at 33(DEGREES), the permissive temperature, but changed to a normal phenotype at 39(DEGREES).^ Two viral proteins were detected in 6m2 cells. A 58,000 dalton protein (P58) was detected at both 33(DEGREES) and 39(DEGREES) and contained only core protein (gag) coded sequences. An 85,000 dalton protein (P85) was detected only at 33(DEGREES) and contained sequences of viral core proteins p15, pp12, and part of p30 as well as protein sequences attributed by peptide mapping to P23 and P38, two candidate viral mouse src (v-mos) gene products. These results provide good evidence that P85 is a gag-mos polyprotein. As expected for a functional mos-gene product, P85 synthesis preceded parameters characteristic of the transformed state, including changes in cell morphology, in the cytoplasmic microtubule complex (CMTC) and in the rate of hexose uptake.^ Other studies were conducted to ascertain the defect which prohibited the synthesis of P85 at 39(DEGREES), the non-permissive temperature. When 6m2 cells were treated with actinomycin D at 39(DEGREES) and shifted to 33(DEGREES), the cells were unable to synthesize P85, but P58 continued to be made. P85 mRNA, active at 33(DEGREES), continued to be translated for two to three hours after shifting to 39(DEGREES) as judged by pulse-labeling experiments. Virus harvested at 33(DEGREES) from ts110 MuSV producer cells packaged both P85 and P58 coding RNAs while virus harvested at 39(DEGREES) was deficient in the amount of P85 coding RNA. Agarose gel electrophoresis of 6m2 cellular RNA showed that RNA harvested at 33(DEGREES) contained the 4.0 and 3.5 kb RNAs. Similar experiments on cells maintained at 39(DEGREES) have detected only the 4.0 kb RNA, suggesting that the 3.5 kb RNA codes for P85. The defect appeared to be in the long term stability of the P85 coding RNA at 39(DEGREES), since, in shift-up experiments (33(DEGREES) (--->) 39(DEGREES)), P85 was translated for only three hours at 39(DEGREES), while P58 was synthesized for at least eight hours. However, at 33(DEGREES) in the presence of actinomycin D, the ratio of P85 and P58 synthesis at hourly intervals was similar throughout a 12 hour period. ^

Dielectrophoresis-based analyte separation and analysis

Dielectrophoresis—the tendency of a material of high dielectric permittivity to migrate in an electrical field gradient to a region of maximum field strength—provides an ideal motive force for manipulating small volumes of biological analytes in microfluidic microsystems. The work described in this thesis was based on the hypothesis that dielectrophoresis could be exploited to provide high-resolution cell separations in microsystems as well as a means for the electrically-controllable manipulation of solid supports for molecular analysis. To this end, a dielectrophoretic/gravitational field-flow-fractionation (DEP/G-FFF) system was developed and the separation performance evaluated using various types and sizes of polystyrene microspheres as model particles. It was shown that separation of the polystyrene beads was based on the differences in their effective dielectrophoretic properties. The ability of an improved DEP/G-FFF system to separate genetically identical, but phenotypically dissimilar cell types was demonstrated using mixtures of 6m2 mutant rat kidney cells grown under transforming and non-transforming culture conditions. Additionally, a panel of engineered dielectric microspheres was designed with specific, predetermined dielectrophoretic properties such that their dielectrophoretic behaviors would be controllable and predictable. The fabrication method involved the use of gold-coated polystyrene microsphere cores coated with a self-assembled monolayer of alkanethiol and, optionally, a self-assembled monolayer of phospholipid to form a thin-insulating-shell-over-conductive-interior structure. The successful development of the DEP/G-FFF separation system and the dielectrically engineered microspheres provides proof-of-principle demonstrations of enabling dielectrophoresis-based microsystem technology that should provide powerful new methods for the manipulation, separation and identification of analytes in many diverse fields. ^

CHARACTERIZATION OF CYTOCHROME P-450 MIXED FUNCTION OXIDASE OF RAT COLON MUCOSA

Non-pregnant, female adult rats pretreated with either phenobarbital (PB) or (beta)-naphthoflavone ((beta)NF) through short-course intraperitoneal injections were shown by sodium dithionite-reduced carbon monoxide difference spectroscopy and NADPH-cytochrome c in vitro assay to contain cytochrome P-450 and NADPH-dependent reductase associated with the microsomal fraction of colon mucosa. These two protein components of the mixed function oxidase system were released from the microsomal membrane, resolved from each other, and partially purified by using a combination of techniques including solubilization in nonionic detergent followed by ultracentrifugation, anion exchange and adsorption column chromatographies, native gel electrophoresis, polyethylene glycol fractionation and ultrafiltration.^ In vitro reconstitution assays demonstrated the cytochrome P-450 fraction as the site of substrate and molecular oxygen binding. By the use of immunochemical techniques including radial immunodiffusion, Ouchterlony double diffusion and protein electroblotting, the cytochrome P-450 fraction was shown to contain at least 5 forms of the protein, having molecular weights as determined by SDS gel electrophoresis identical to the corresponding hepatic cytochrome P-450. Estimation of total cytochrome P-450 content confirmed the preferential induction of particular forms in response to the appropriate drug pretreatment.^ The colonic NADPH-dependent reductase was isolated from native gel electrophoresis and second dimensional SDS gel electrophoresis was performed in parallel to that for purified reductase from liver. Comparative electrophoretic mobilities together with immunochemical analysis, as with the cytochrome P-450s, reconstitution assays, and kinetic characterization using artificial electron acceptors, gave conclusive proof of the structural and functional homology between the colon and liver sources of the enzyme.^ Drug metabolism was performed in the reconstituted mixed function oxidase system containing a particular purified liver cytochrome P-450 form or partially pure colon cytochrome P-450 fraction plus colon or liver reductase and synthetic lipid vesicles. The two drugs, benzo{(alpha)}pyrene and benzphetamine, which are most representative of the action of system in liver, lung and kidney, were tested to determine the specificity of the reconstituted system. The kinetics of benzo{(alpha)}pyrene hydroxylation were followed fluorimetrically for 3-hydroxybenzo{(alpha)}pyrene production. . . . (Author's abstract exceeds stipulated maximum length. Discontinued here with permission of author.) UMI ^