On the electrophoretic mobility of biological cells

A theoretical study has been performed on the electrophoretic mobility of a large colloidal particle with a surface charge layer as a model for biological cells. An approximate mobility expression is derived which is applicable to the general case of an arbitrary distribution of membrane-fixed charges. This expression consists of two terms: the first is a weighted average of potentials over the surface charge layer, while the second is that of the volume density of membrane-fixed charges and does not depend on the electrolyte concentration. At high electrolyte concentrations (corresponding to physiological conditions), where the potentials are very low owing to the shielding effects of electrolytes, the first term diminishes so that the mobility is determined mainly by the second term. This means that a particle with zero surface potential can exhibit a non-zero mobility, in contrast to the prediction from the classical Smoluchowski theory. Comparison is also made with the theory of Hermans and Fujita for the electrophoresis of polyelectrolytes.     

An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5( 1.00) allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model.     

Phosphoglucomutase polymorphism in the oriental fruit fly, Dacus dorsalis (Diptera, Tephritidae) from Peninsular Malaysia

Seven natural populations of Dacus dorsalis were analysed for phosphoglucomutase by means of horizontal starch-gel electrophoresis. The electrophoretic phenotypes were governed by four codominant Pgm alleles. The commonest allele in all the seven population samples was PgmB which encoded an electrophoretic band with intermediate mobility. The distributions of PGM phenotype were in accordance with Hardy-Weinberg expectations. There was geographic variation in the distribution of Pgm alleles.     

A multi-locus continuous-time selection model

Summary A continuous time selection model is formulated for a diploid monoecious population with multiple alleles at each of an arbitrary number of loci, incorporating differential fertility and mortality as well as arbitrary mating and age structure. The model is simplified in the case of age-independence and for the case of a stable age distribution. The age-independent model is examined in detail for the special case of multiple alleles at each of two loci. This model is analyzed under the assumptions of random mating and additive fertilities, with close attention given to the behavior of the system with respect to Hardy-Weinberg proportions and linkage equilibrium.M. M. was supported by a U.S. Public Health Service training grant (Grant No. GM780).     

Variation in Activities of Amylase Allozymes Associated with Chromosome Inversions in DROSOPHILA PSEUDOOBSCURA, D. PERSIMILIS and D. MIRANDA

Different electrophoretic alleles of amylase show associations with particular chromosome 3 inversions in D. pseudoobscura and D. persimilis. Relative adult amylase activities were compared in 37, 37 and 10 strains of D. pseudoobscura, D. persimilis and D. miranda, respectively. Strains carrying the same electrophoretic allele were compared by crossing these lines individually to a reference strain carrying a different electrophoretic mobility allele. This procedure allows comparisons among species, inversions, electromorphs and strains for genetic variation in amylase activity. F2 analysis established that the activity variation co-segregates with the structural amylase locus. This type of variation could be due to either structural gene differences or differences in closely linked, cis-acting regulatory regions. Variation has been detected among and within electrophoretic mobility classes. Moreover, this variation is clearly nonrandom and reveals more of the genetic structure associated with the chromosomal inversion phylogeny of D. pseudoobscura and D. persimilis. ----Some of the findings are: (1) Similar electromorphs in D. pseudoobscura and D. persimilis usually show different activities. These species show nearly complete differentiation of amylase alleles, based on activities. (2) D. persimilis has the broadest range of variation in amylase activity, about four-fold between the highest and lowest alleles. D. pseudoobscura and D. miranda are also polymorphic for activity, but have more constrained ranges of variation. D. miranda alleles show on the average about four times the activity of D. pseudoobscura alleles. (3) Some association of electrophoretic mobility and activity has been found. Alleles 1.09 of D. persimilis, as well as 1.43 and 1.55 of D. miranda, have relatively high activity. It may be that these high activity alleles are part of an adaptation to cooler habitats. (4) Within electrophoretic classes, associations of activities with inversions have been found. These are especially strong in D. persimilis. The 1.00 alleles in the ST, KL, MD and WT inversions, the 0.92 allele in the ST and MD inversions and the 1.09 allele in the WT and KL inversions have levels of activities that depend upon the arrangement in which they are located. These results demonstrate that suppression of recombination in inversion heterokaryotypes can result in extensive genic divergence between inversions.     

Exact solution of the multi-allelic diffusion model

We give an exact solution to the Kolmogorov equation describing genetic drift for an arbitrary number of alleles at a given locus. This is achieved by finding a change of variable which makes the equation separable, and therefore reduces the problem with an arbitrary number of alleles to the solution of a set of equations that are essentially no more complicated than that found in the two-allele case. The same change of variable also renders the Kolmogorov equation with the effect of mutations added separable, as long as the mutation matrix has equal entries in each row. Thus, this case can also be solved exactly for an arbitrary number of alleles. The general solution, which is in the form of a probability distribution, is in agreement with the previously known results. Results are also given for a wide range of other quantities of interest, such as the probabilities of extinction of various numbers of alleles, mean times to these extinctions, and the means and variances of the allele frequencies. To aid dissemination, these results are presented in two stages: first of all they are given without derivations and too much mathematical detail, and then subsequently derivations and a more technical discussion are provided.     

The Genetics of DROSOPHILA SUBOBSCURA Populations. Xvii. Further Genic Heterogeneity within Electromorphs by Urea Denaturation and the Effect of the Increased Genic Variability on Linkage Disequilibrium Studies

Urea denaturation of allozymes was used to provide finer resolution of allelic states within classes of different electrophoretic mobility. This method gives perfectly repeatable results. About 170 isogenic strains for the O chromosome of Drosophila subobscura, derived from two natural populations, were constructed. Their gene arrangements were studied, as well as eight polymorphic genes located on the O chromosome (Est-5, Odh, Ao, ME, Xdh, Lap, Pept-1 and Acph). Crosses performed indicate that differences in urea sensitivity are genetically controlled by the same genes that control electrophoretic mobility. Twice as many alleles have been detected in comparison to the usual electrophoretic method. However, the effective number of alleles did not increase considerably.Studies of linkage disequilibria, by taking into account the finer resolution of allelic states, gave results nearly identical with those obtained in studies where the usual electrophoretic method was used. Although the power of the test is diminished, the absence of genic associations seems to indicate that there are no hidden linkage disequilibria in electrophoretic studies (because of consolidation effects of real alleles into few electromorph classes). The paucity of linkage disequilibria would indicate that there are no epistatic interactions such as those suggested in the model of Franklin and Lewontin (1970).     

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.     

Continuous selective models

Neglecting age-structure, but taking into account matings with differential fertility in Mendelian reproduction, continuous selective models are formulated for a single locus with an arbitrary number of alleles, with or without distinguishing the sexes, and for two alleles at each of two loci in a monoecious population. In each case, without restricting the mating system, differential equations are derived for the genotypic frequencies, and the validity of the customary Malthusian-parameter differential equations for the gametic frequencies is established. Particular attention is devoted to the conditions for Hardy-Weinberg proportions under random mating. For multiple alleles at a single locus in a monoecious population, exact solutions are obtained for the following three Hardy-Weinberg models: gametic selection, no dominance, and the same selective effect for all alleles but one. The last scheme includes, as special cases, a completely dominant or recessive distinguished allele, and arbitrary selection with only two alleles. Two single-locus assortative mating patterns are analyzed for a monoecious organism using the general formalism. One of these has an arbitrary number of alleles, all the genotypes being distinguishable, while the other involves two alleles, one of which is completely dominant to the other.     

Heterologous expression and characterization of the human R-ras gene product.

We directly expressed human R-ras 23,000-dalton protein (p23) cDNA in Escherichia coli under the control of the trp promoter. GTP-dependent phosphorylation of a p23 threonine 85 substitution mutant was observed. This result is in direct analogy to the autokinase activity of H-ras and K-ras threonine 59 substitution mutants. Normal p23 protein was detected in the human fibrosarcoma cell line HT1080 by immunoprecipitation with rabbit antibodies raised against an E. coli-expressed R-ras fusion protein. The R-ras p23 protein was found to be 3H labeled in the presence of [9,10(n)-3H]palmitic acid and is associated with the P100 membrane fraction of HT1080 cells. These data suggest that human R-ras p23 has biochemical properties very similar to those of the p21 products of the H-, K-, and N-ras proto-oncogenes. We constructed an R-ras minigene and engineered the expression of normal and mutant alleles from the simian virus 40 early region promoter. Normal and mutant R-ras gene products were authenticated by transient expression in COS-7 cells and immunoprecipitation. The valine 38-substituted R-ras p23 displayed reduced electrophoretic mobility. R-ras p21-like proteins, made by eliminating the first 26 R-ras codons, displayed evident mobility differences between the pro form and mature form, along with a valine 12 substitution-dependent change in electrophoretic mobility. Rat-1 fibroblasts were transfected with normal and mutant R-ras alleles and normal and activated H-ras alleles. Unlike the human T24 bladder oncogene-encoded p21, mutant R-ras alleles do not cause monolayer focus formation or growth in soft agar of rat fibroblasts.     

Iminoxyle Radical with Isocyanide as a Spin Label for Haemoproteins

BULMER¹ has analysed data on multiple allelic series of genes determining enzymes and has shown that if the alleles are arranged in order of electrophoretic mobility, there is a marked tendency for the rare alleles to occur at the beginning or the end of a sequence and for the common alleles to occur in the middle of a sequence.     

Evaluation of the Stepwise Mutation Model of Electrophoretic Mobility: Comparison of the Gel Sieving Behavior of Alleles at the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA

Seven alleles at the esterase-5 locus of Drosophila pseudoobscura appear approximately uniformly spaced on 5% acrylamide gels. Such stepwise "ladders" in mobility have been used to argue for the charge-state model of electrophoretic mobility. To evaluate this interpretation, flies of the seven strains were examined in replicate electrophoresis on polyacrylamide gels of differing pore size, permitting estimation of the relative contributions of charge and of size/conformation to electrophoretic mobility. Six of the seven strains examined proved to be heterogeneous, containing multiple variants that migrate to similar positions on 5% acrylamide gels. In the one strain genetically analyzed to date, the hidden variants segregate in crosses. A total of fourteen variants are detected by this gel sieving analysis, many of them involving apparent conformational differences. Thus, protein properties in addition to net charge appear to play an important role in determining the degree of mobility difference between alleles. Examining estimates of free mobility, uniform charge differences are the rule within conformational classes. However, the superposition of conformational heterogeneity renders interpretation of mobility spacing solely in terms of such charge differences inappropriate.     

Multiple alleles and low variability of glycerol-3-phosphate dehydrogenase in Aedes albopictus (Diptera: Culicidae) from Peninsular Malaysia

1. A total of 8 samples from three natural populations and a laboratory strain of Aedes albopictus were analysed for glycerol-3-phosphate dehydrogenase phenotypes by means of horizontal starch-gel electrophoresis. 2. The electrophoretic phenotypes were governed by three codominant Gpd alleles. 3. There was low variability, with the heterozygosity in the variable samples ranging from 0.02 to 0.12. 4. The commonest allele in all the population samples was GpdB which encoded an electrophoretic band with intermediate mobility. 5. There was no temporal or spatial variation.     

Significance of electrophoretic mobility distribution to bacterial transport in granular porous media

A method is developed to obtain the electrophoretic mobility distribution of colloidal particles by microelectrophoresis. The results demonstrate that for small particles (< 1 microm), the experimental mobility distribution must be deconvoluted to remove the effect of the random Brownian motion so that the electrophoretic mobility distribution can be obtained. For bacteria-sized particles (on the order of 1 microm or larger), the random Brownian motion is not significant, and the experimental mobility distribution represents the electrophoretic mobility distribution. The significance of the electrophoretic mobility distribution to bacterial transport is demonstrated through comparison between experimental and theoretical values of collision efficiency. Using the extended Derjaguin-Landau-Verwey-Overbeek (DLVO) theory, the electrophoretic mobility distribution of bacteria is transformed to the distribution of collision efficiencies. For strain Comamonas sp. DA001, the predicted collision efficiency values span orders of magnitude, indicating that variation of surface charge density in a monoclonal bacterial population is a cause for the orders of magnitude variation of experimentally determined collision efficiencies. However, despite the fact that the predicted and experimental alpha distributions overlap, the match is not adequate. This inadequacy is ascribed to inability to probe heterogeneity of bacterial surface hydrophobicity, and the inability of the DLVO theory to quantitatively model particle deposition.     

Genetic analysis of an electrophoretic variant for the chloroplast-associated form of Cu/Zn superoxide dismutase in sunflower (Helianthus annuus L.)

Polyacrylamide gel electrophoresis of water-soluble proteinsfrom sunflower (Helianthus annuus L.) cotyledons, followed byspecific staining for superoxide dismutase activity, discriminated,according to their electrophoretic mobility, two distinct achromaticbands for Cu/Zn superoxide dismutase. Zymograms of proteinsfrom isolated chloroplasts showed that the chloroplast-locatedCu/Zn superoxide dismutase (Cu/ZnSOD_Chl) migrated faster inthe SOD activity-stained gels. An electrophoretic variant pattern,whose mobility is lower than the control pattern, was identifiedin the ABA-deficient mutant w-1. The variant is coded by a nucleargene with two codominant alleles. Key words: Sunflower, Helianthus annuus L., ABA-deficient mutant, electrophoretic isozyme variant, superoxide dismutase     

Comparative electrophoretic properties of histones from trout and chicken erythrocytes and calf thymus at different concentrations of EDTA]

An introduction of EDTA into an electrophoretic system was found to cause specific changes in the histone distribution patterns. The electrophoretic mobility of histones H3, H2b and H2a from three evolutionally unrelated sources (trout and chicken erythrocytes and calf thymus) is increased and that for histones H1 and H5 is decreased with respect to histone H4. In general the decrease of electrophoretic mobility of the histones in the presence of EDTA is correlated with the content of basic amino acids in these histones. The effect observed can be used from electrophoretic analysis of histones.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

Classification of the ceruloplasmin locus in cattle]

The object of this study was the polymorphism of the ceruplasmin locus in different separate herds of cattle of the Pinzgan and the Red Polish breeds. The investigations carried out corroborated the hypothesis of the triallelic nature of this locus, which expands our knowledge and possibilities in this direction. The frequency of the alleles A, B and C in Red Polish cattle (n = 333) was 0,725; 0,026 and 0,249 respectively, being 0,920; 0,009 and 0,071 in the Pinzgan cattle (n = 369). An assumption is made about the greater value of proteins of a relatively high electrophoretic mobility as compared to those of a relatively low electrophoretic mobility.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

Changes in electronegativity of the cell surface during the development of the cell surface during the development of the cellular slime mold, Dictyostelium discoideum

A microelectrophoretic method was applied to determine electrophoretic mobility of the amoebae of the cellular slime molds at various stages of development. The vegetative amoebae were negatively charged and their electrophoretic mobility remained unchanged as long as they fed. After the cessation of feeding mobility of the amoebae decreased gradually in the interphase. Proteolytic enzymes and EDTA had no effect on mobility of the amoebae right after finishing feeding. On the contrary, mobility of the amoebae in the middle interphase was increased by treatment with proteolytic enzymes, lipase, and EDTA. EDTA and pronase showed an additive effect on the increase in mobility, and the increased mobility was almost the same as that of the vegetative a-moebae. These results indicate that the decrease in mobility during the interphase is due to the accumulation of a substance(s) (probably lipoprotein) on the cell surface. The cells disaggregated from the migrating slugs showed lower mobility than the cells obtained from the aggregation centers. The latter had still lower mobility than the interphase amoebae. These changes in electrophoretic mobility were discussed in relation to corresponding changes in adhesiveness.