George W. Nace

Staining acidic phosphoproteins (phosvitin) in electrophoretic gels

The principal phosphoglycoproteins of avian and amphibian egg yolk, known as phosvitins, have been examined extensively in developmental studies of estrogenic induction of protein synthesis (1) and, more recently, as substrates for protein kinases which phosphorylate the basic nuclear proteins (2,3). Phosvitin has few aromatic and basic amino acids (4) which can be detected by uv absorbance or by conventional anionic protein stains following electrophoresis. In addition, the high negative-charge density of clustered phosphorylserine residues (2) may prevent staining by charge repulsion of anionic dyes. Dilute solutions of cationic dyes like toluidine blue and acridine orange have been employed (5,6), but, in our experience, they penetrate gels slowly and also stain the residual negative charges of most electrophoretic matrices, including agarose, starch, and even polyacrylamide. The only specific method for localizing phosphoproteins in polyacrylamide gels requires several time-consuming steps to liberate orthophosphate by basic hydrolysis, to form an insoluble phosphomolybdate complex, and, finally, to stain this complex with methyl green dye (7). The exceptional affinity of contiguous phosphorylserines for trivalent metal ions (8) can, however, be exploited to detect phosvitin in gels. This strategem may also provide an alternative method for visualizing similar acidic phosphoproteins which have now been identified in brain, spermatozoa, and adrenal medulla (9,lO).

Mixing device for generating simple chromatographic gradients

Abstract A linear gradient device consisting of two concentric reservoirs of equal cross-sectional area, in which the initial and limiting eluent solutions are maintained in hydrostatic equilibrium, may be constructed from several combinations of commercial acrylic resin cylinders. By the use of nesting cylinders, the areas of the reservoirs may be reduced independently, allowing an assortment of nonlinear gradients to be produced by simple adjustment of the area ratio of the two reservoirs.

Inheritance of Enzymes and Blood Proteins in the Leopard Frog, Rana pipiens: Three Linkage Groups Established

Individuals from natural populations of the leopard frog, Rana pipiens, were analyzed for electrophoretic differences in blood proteins and enzymes from an amputated digit. The proteins examined represent products of 72 loci. Presumptive heterozygotes at multiple loci were selected for experimental crosses. Mendelian inheritance of 18 protein variations were demonstrated in the offspring. Tests for linkage or independent assortment were performed for 75 locus pairs. Three linkage groups were established. Linkage group 1 contains two loci, aconitase-1 (Acon1) and serum albumin (Alb), with a 19% recombination frequency between them. Linkage group 2 contains four loci, glyoxalase (Gly), acid phosphatase-1 (Ap1), acid phosphatase-2 (AP2), and esterase-5 (Est5). The data show the relationships Gly-21.1%-AP1-0%-AP2-6.3%-Est5, and Gly-25.6%-Est5. Linkage group 3 consists of four closely linked esterase loci. The data, Est1-5.1%-Est6, Est6-1.8%-Est10-1.9%-Est4 and Est6-3.0%-Est4, do not establish a complete order but suggest that Est10 is between Est4 and Est6. These results, with data demonstrating apparent independent assortment of 67 other locus pairs, provide a foundation for establishing the frog genetic map.

Development of Biologically Defined Strains of Amphibians

Throughout this Symposium (1) two threads of thought are implicit. The iirst is that the amphibian is a desirable organism on which to conduct tumor and virus research, but that living amphibian material, either in culture or as the whole animal, must be brought under more adequate biological control. Reference has repeatedly been made to the use of wild amphibians in a variety of experimental situations. In no case has reference been made to the nutritional or health status of the animals involved. The only attempts at genetic definition have been by reference to karyotyping, with the exception of work with some urodeles which have been maintained in laboratory colonies for significant periods. It is evident from the work of Moore (2) and McKinnell (3) that the biology of Rana pipiens from closely adjacent populations may differ markedly. Important genetic differences among animals from different commercial shipments must be expected. The second line of thought is that viruses are somehow associated with the development of the Lucke renal adenocarcinoma. Only the approach developed by Tweedell (4) and utilized by Mulcare (5) has routinely resulted in the induction of tumors by inoculation of a virus-containing fraction. Tumors have been induced in other cases (6) but the incidence has been below that desirable for routine analysis of the biology of this tumor. Granoff (7) has specifically indicated the failure to induce tumors in frogs with cultured agents isolated from tumor-bearing frogs.

Sources of Amphibians for Research

The program of the Amphibian Facility of The University of Michigan has been described in the reports listed in the bibliography. Nace (1968) contains an overall description; the other papers cited describe specific features. Nace (1970) reproduces the information forms which record data on demography, genetics, and pathology of the animals in the Amphibian Facility. It also includes tabular information on the

Dark pigment variants in anurans: classification, new descriptions, color changes and inheritance

Dark pigment variants are described in four species of frogs, Rana pipiens, R. clamitans, R. sylvatica and Ptychadena mascareniensis. Guidelines are proposed for recognition and description of dark variants by field collectors. A classification scheme is proposed to categorize the many phenotypic types seen among dark frogs. Breeding experiments demonstrated that the dark gene was sometimes expressed in the heterozygous condition making it a dominant gene but that penetrance was so low that in most instances it appeared to act as a recessive. Color change sometimes occurred in captive dark frogs. These were always increases in normal pigmentation rather than of dark pigmentation.

Tissue localization of multiple forms of enzymes.

The species specificity of enzymes and the organ specificity of isozyme combinations is no longer questioned. The attributes of tissue, cellular and sub-cellular specificity of enzymes, however, arc quite uncertain. Organ specificity could, and probably does, arise as a result of any of several modes of enzyme distribution at these lower levels of organization. Thus, organ specific isozyme patterns may arise if each isozyme is characteristic of a given tissue or cell type, since organs differ from each other by the ratio and selection of their component tissue and cell types. Alternatively, these patterns may arise if more than one isozyme is found in each tissue or cell type. These possibilities imply quite different mechanisms for the genetic control of isozymes. If one isozyme corresponds to one cell type, only a single gene-to-protein sequence need be functional in a given cell; and the different isozymes of different cell types could represent either the modulation of the action of the single locus by subsidiary genetic or cytoplasmic factors, or the action of different loci. If, on the other hand, a cell contains more than one isozyme, either a single locus must form a precursor substance whose final forms are determined by extra-chromosomal factors in the sub-cellular environment (S. Allen, 1961; Levinthal et al., 1962), a multiplicity of genetic loci must function in a given cell type as implied by the polymerization concept (Cahn et al., 1962), or a combination of these mechanisms must be operative. A t the sub-cellular level, any of several possibilities of isozyme distribution would be compatible with the several possible modes of isozyme distribution and production at the tissue and cellular levels. For example, a given isozyme might be unique to a given type of sub-cellular structure, or it might be localized on different sub-cellular strwtnres in different cell types. Current data do not allow selection among the various possibilities outlined above or were obtained by techniques that are not generally applicable. Mechanical separation, in quantity, of the tissues comprising complex organs lacks the necessary precision and does not seem to have been attempted. Pure line culture of various tissue or cell types is possible. Tsao

Frog lysozyme. II. Isozymes of lysozyme during the larval development of the leopard frog, Rana pipiens

Abstract Eight isozymes of lysozyme were found differentially distributed among six developmental stages of Rana pipiens . Qualitative changes during early development involved a progressive loss of the more basic isozymes which then reappeared between metamorphosis and maturity. Though the egg contained five isozymes, only two were present by early metamorphosis including one not found in the egg. By metamorphic climax, the four isozymes in the egg were regained and one additional form appeared. By maturity, two less basic forms appeared giving a total of eight isozymes. From hatching to early metamorphosis, lysozyme units per animal increased, but lysozyme units/mg dry weight remained unchanged. Both lysozyme units per animal and units/mg dry weight increased sharply towards the end of metamorphosis.

A Developmentally Significant Antigen in the Surface of Frog Embryo Cells

Abstract Data demonstrating the role of yolk pseudoglobulin in maintaining the integrity of frog neurula cells in hanging drop cultures were recalled. Evidence was presented to demonstrate that the cytolysis of these cultured cells by antineurula sera was preceded by in situ lysis of yolk platelets. It was shown that washed yolk platelets directly exposed to the cytolytic antisera as well as to other antisera were not lysed. This led to the conclusion that the cytolysis of the cultured cells must be attributed to the action of the antisera on cell membrane antigens with pseudoglobulin specificity rather than to the passage of antibody into the cells.