Arylsulfatase A: Relationship of genotype to variant electrophoretic properties

Previous work has shown that specific electrophoretic variants of arylsulfatase A occur more frequently among alcoholic patients than among psychiatric and normal controls. The present study sequenced the gene for two of these electrophoretic variants, IIIa and IIIb. Both contain an A-to-G transition corresponding to substitution of Asn₃₅₀ by Ser, with the resulting loss of anN -glycosylation site. The difference in electrophoretic mobility of their gene products is due to a mutation in the IIIb gene resulting in the replacement of Arg₄₉₆ by His. Evidence is presented that individuals possessing either of two other electrophoretic variants, Va and Vb, are heterozygous for a normal ASA allele and either a IIIa or IIIb allele, respectively. Thus, the relationship between the phenotype of the electrophoretic banding patterns, IIIa, IIIb, Va, and Vb, and their corresponding genotypes has been elucidated.     

小麦抗锈变异体的RAPD分子验证

将长穗偃麦草DNA导入普通小麦甘麦8号,在其后代出现广泛变异,并从中选育出两个优良的抗条锈病的姊妹变异体。本实验以原供体和受体作为对照,对两个变异体进行了RAPD分子验证,其主要结果为：两个变异体的大多数引物扩增产物带型类同于受体,而与供体带型差异显著;在81个引物中有2个引物检测出了DNA的多态生,主要表现为变异体90001-17中1条带活性不仅超过受体,而且也远超过90001-1的相应带活性,同时两个变异体都出现了1条供体和受体都没有的新带,分子量约为1044bp;另一引物扩增后,变异体90001-17和90001-1分别出现了2条和1条特异性带,分子量约为1073bp和1020bp,此外在两个变异体阳极端带明显被激活,而受体中1条分子量约为968bp的带在变异体中活性降低或消失等。从而表明抗锈变异体9001-17和90001-1的形成是供体的DNA片段进入受体基因组的结果。     

Hereditary erythrocyte pyruvate-kinase (PK) deficiency and chronic hemolytic anemia: Clinical,genetic and molecular studies in six new Spanish patients

Summary Clinical, familial and biochemical studies from six unrelated Spanish patients with hereditary hemolytic anemia and erythrocyte pyruvate-kinase (PK) deficiency are described. A remarkable molecular heterogeneity of mutant PK variants involving kinetic properties, molecular stability or electrophoretic mobility is demonstrated. In two patients whose PK variants showed abnormal electrophoretic pattern, and strongly aberrant kinetic properties, a chronic hemolytic anemia assciated with several other clinical manifestations of chronic hemolysis occurred. In patients whose PK variants showed less abnormal kinetic and electrophoretic characteristics, there was only moderate or mild hemolytic anemia. One patient's PK variant with no obvious kinetic or electrophoretic alterations, showed a markedly decreased heat stability with severe diminution of antigenic concentration of the enzyme. This patient presented a spectacular clinical and hematological improvement after splenectomy.The purpose of the present study is to describe six new PK variants of Spanish origin. In addition, an attempt is made to find relationships between molecular abnormalities of mutant PK variants and the severity of hemolytic anemia, in these patients. The possible role of some kinetic alterations, such as fructose diphosphate (FDP) activation or ATP inhibition of PK variants, in the clinical manifestations of chronic hemolysis is also suggested.     

A "disproportion" between the frequency of rare electropmorphs and enzyme deficiency variants in Amerindians

Our previous studies have revealed a higher frequency of nonpolymorphic electrophoretic variants in blood samples from Amerindians than in similar samples from Caucasians and Japanese. Our present study finds, by contrast, that the frequency of deficiency variants of 11 erythrocyte enzymes, sampled in nine Amerindian tribes of Central and South America, is essentially the same (1.5/1,000 determinations) as in Caucasians or Japanese. Possible explanations of the elevated frequency of mobility variants in the tropical-zone/ unacculturated populations include: higher mutation rates resulting in both electrophoretic and activity variants in Amerindians but increased selection against deficiency variants in the Amerindians, or comparable mutation rates in both populations coupled with a greater probability of a mobility variant attaining a relatively high frequency among the Amerindians.     

Modification of alkaline phosphatases by treatment with glycosidases

The tissue-specific variants of alkaline phosphatase that are characteristic of human liver and bone are believed to possess identical protein cores; nevertheless, they differ in certain properties such as electrophoretic mobility and stability to heat. Their electrophoretic mobilities are modified by digestion with various glycosidases. Furthermore, the difference in heat stability between them is reduced by treatment with a glycosidase preparation from Trichomonas foetalis. These results are consistent with the view that these enzyme variants differ only in their carbohydrate moieties.     

An electrophoretically cryptic alcohol dehydrogenase variant in Drosophila melanogaster. I. Activity ratios, thermostability, genetic localization and comparison with two other thermostable variants

The alcohol dehydrogenase (ADH) variant ADH-FCh.D. has a secondary alcohol/primary alcohol activity ratio characteristic of ADH-S although it has an electrophoretic mobility inseparable from ADH-F. ADH-FCh.D. is distinguished from these two common ADH variants by being much more thermostable. Genetic analysis suggests tht ADH-FCh.D. is specified by an allele at the Adh locus. Biochemical comparisons show that ADH-FCh.D. has the same electrophoretic mobility, activity ratio and thermostability as the two other heat-resistant variants which have been reported, ADH-F71K in Europe and ADH-Fr in North America. The geographically widespread distribution of a thermostable ADH variant within the ADH-F electrophoretic class indicates that it should be considered in attempts to explain the Adh polymorphism in natural populations.     

Hemolytic anemia due to pyruvate kinase deficiency: characterization of the enzymatic activity from eight patients.

We have studied the red cell pyruvate kinase (PK) variants from eight patients representing five families with pyruvate kinase deficiency-associated hemolytic anemia. The kinetic properties, electrophoretic mobilities, and immunological reactivity with anti-normal red cell pyruvate kinase were determined. The patients differ in the severity of their clinical condition and in the molecular properties of their red cell pyruvate kinase variants. The most seriously affected patient (PK Beaverton) has no electrophoretically demonstrable red cell isozymes. The activity present is due to the M2 isozyme, however red cell isozyme can be detected immunologically. PK Molalla and PK Lake Oswego are thermolabile variants with normal kinetic parameters. PK Molalla, in addition, has altered electrophoretic mobility. PK Multnomah and PK Milwaukie have decreased affinity for the substrate phosphoenolpyruvate, and PK Multnomah also has altered electrophoretic mobility. PK Coos Bay shows electrophoretic variation and a slightly decreased affinity for phosphoenolpyruvate consistent with an increased modulating effect of fructose-1,6-diphosphate.     

Comparison of acid phosphatase ACP1 variants by isoelectric focusing and conventional electrophoresis: identification of three new alleles, ACP1*N, ACP1*P and ACP1*S

Three new alleles of human red cell acid phosphatase (ACP1) have been identified by comparison with previously reported variants using three different electrophoretic techniques. Family data are available on all the variants and show genetic transmission of the rare alleles ACP1*N, ACP1*P and ACP1*S. Further evidence of a rare allele demonstrating reversed 'A' activity is also described. The report documents the need to use several electrophoretic techniques to characterize new or rare variants.     

Estimating total genic diversity in the house mouse

In a survey of variation in both electrophoretic charge and thermostability at 14 structural loci in 40 strains of Mus musculus, 27 electromorphs (polypeptides differing in electrophoretic charge) and 20 thermomorphs (polypeptides differing in thermostability) were found. Electrophoresis detected 11 new variants within thermomorphs, and the heat denaturation technique detected four new variants within electromorphs. From these data, and making the assumption that both techniques are independent of each other, it is estimated that the actual total number of alleles at the 14 loci is 53, or an average of 3.79 per locus (1.96 per electromorph), and that electrophoresis apparently detects one-third of the variants, thus describing about 50% of the alleles at structural gene loci in the house mouse.     

Electrophoretic variability in a total of 11 structural sites in murine rodents (Muridae, Rodentia)

Forty-six rodent species of the Muridae family were submitted to sequential electrophoresis for the study of 11 protein loci, using 12 buffer systems which differed in pH and ionic composition. The complete set of electrophoretic conditions yielded 135 variants of which 68 were detected through a single condition. The twofold increase of revealed variants was essentially limited to intergeneric comparisons because few additional variants within genera were revealed, despite the use of several buffers. These results show that estimation of the degree of genetic differentiation among taxa at a higher level than that of the genus by standard electrophoretic data may, although of current use, lead to erroneous results.     

Inherited deficiency of the sixth component of complement: a silent or null gene

Four families have been studied, some members of which have inherited deficiency of the sixth component of complement. The genetically determined electrophoretic variants of C6 were evaluated in all family members. Seven individuals were found who did not have the variant found in the serum of the parent from whom they inherited the deficiency. It is inferred that the isolated low levels of C6 in these individuals results from the heterozygous state of a normal C6 variant gene and a silent or null C6 gene; the genes determining electrophoretic variants and the low serum levels of C6 are allelic.     

Isoallele Frequencies in Very Large Populations

The frequencies of electrophoretically distinguishable allelic forms of enzymes may be very different from the corresponding frequencies of structurally distinct forms, because many sequence variants may have identical electrophoretic charge. In large populations such frequencies will be determined largely by the number of amino acid sites that are free to vary. The number of distinguishable electrophoretic variants will remain fairly small. Beyond some limiting size, no further effect of population size on allele frequencies is expected, so isolated large populations will have closely similar allele frequencies if polymorphism is due largely to mutation and drift. The most common electrophoretic alleles are expected to be flanked by the next most common, with the rarer alleles increasingly distal. Neither strong selection nor mutation/drift interpretations of enzyme polymorphism are yet disproven, nor is any point between these extremes.     

IN VIVO Function of Rare G6pd Variants from Natural Populations of DROSOPHILA MELANOGASTER

From 1981 to 1983, 15,097 X-chromosomes were genetically extracted from a number of North American populations of D. melanogaster and were electrophoretically screened for rare mobility and activity variants of glucose-6-phosphate dehydrogenase (G6PD). Overall, 13 rare variants were recovered for a frequency of about 10^-3. Eleven variants affect electrophoretic mobility and are apparently structural, and two variants exhibit low G6PD activity. One low activity variant is closely associated with a P-element insertion at 18D12-13—all of the variants were subjected to the previously described genetic scheme used to identify relative in vivo activity differences between the two common electrophoretic variants associated with the global polymorphism. Most of the rare variants exhibit apparent in vivo activities that are similar to one or the other of the common variants, and these specific rare variants appear to be geographically widespread. Several variants have significantly reduced function. All of the variants were measured for larval specific activity for G6PD as a first measure of in vitro activity. It appears that specific activity alone is not a sufficient predictor for G6PD in vivo function.     

Electrophoretic Variants of Phosphoglucomutase in Saccharomyces Species

Strains of Saccharomyces cerevisiae and species with which S. cerevisiae is interfertile display a characteristic pattern of electrophoretic variants of phosphoglucomutase (PGM) consisting of a major component and one or two minor components, all of which migrate toward the cathode. The patterns are consistent with an earlier finding that two unlinked genes, one of which has two known alleles, determine the synthesis of PGM in S. cerevisiae. The PGM patterns of strains of S. fragilis, S. lactis, and S. marxianus, species thought to be closely related to each other and only distantly related to S. cerevisiae, also displayed a characteristic pattern of PGM variants, but it was quite different from that of S. cerevisiae. In these species five or six electrophoretic variants could be detected, all of which migrated toward the anode. We interpret the differences in the PGM variants of the two groups of species as a reflection of differences in genetic composition which have arisen in two phylogenetically distinct groups that have become sexually isolated from each other.     

Electrophoretic karyotype polymorphism of sibling species of the Paramecium aurelia complex

Variability of karyotypes is one of the main mechanisms of speciation in organisms. Electrophoretic karyotypes of the macronucleus (MAC) obtained by pulsed-field gel electrophoresis were compared for 86 strains of all 15 sibling species of the Paramecium aurelia complex in order to determine if karyotype differences corresponded to biological species boundaries. Because the electrophoretic karyotype of the MAC reflects indirectly the frequency and distribution of fragmentation sites in the micronuclear (MIC) chromosomes, any change in MAC electrophoretic karyotype may be a marker of certain chromosomal mutations in the MIC. Thirteen main variants of electrophoretic MAC karyotypes were observed in this species complex. Ten of them appeared to correspond to biological species, while the three other variants characterized several species each. Intraspecific polymorphism was observed for several species: in some cases a certain variant of MAC karyotype was specific for all strains from the same part of the world. Distribution of the MAC karyotype variants along molecular phylogenetic trees of the P. aurelia complex shows that isolation of each species or group of species of this complex was accompanied by divergence in the molecular organization of the genome.     

Studies of Esterase-6 in DROSOPHILA MELANOGASTER. II. the Genetics and Frequency Distributions of Naturally Occurring Variants Studied by Electrophoretic and Heat Stability Criteria

Measurements of the electrophoretic mobility and thermostability of esterase-6 allozymes have been used to determine the amount of allelic variation at the esterase-6 locus in Drosophila melanogaster. We studied 398 homozygous lines obtained from four natural populations. Use of a spectrophotometric assay for esterase-6 activity has allowed precise quantitation of heat-stability variants. Using these methods, eight putative alleles were detected within the two most common electrophoretic classes. Analyses of F1 and F2 progeny show that the behavior of stability variants is consistent with the hypothesis that this variation is due to allelic variation at the Est-6 locus. Analyses of the gene-frequency distributions within and between populations show (1) that observed allele-frequency distributions do not deviate significantly from those expected for neutral variants, and (2) that there is little evidence for an increase in apparent divergence of the different populations at the genotypic or phenotypic levels when the additional variation detected is considered. These findings suggest that gene-frequency analysis alone is unlikely to resolve the question of the selective significance of allozyme variation.     

Molecular Basis of Transferrin Polymorphism in Goldfish (Carassius auratus)

Transferrin (TF) polymorphism was investigated in a color variety of goldfish (Carassius auratus), and its molecular basis analyzed. Three TF variants (A1, A2 and B1) were identified from an inbred strain of the goldfish, of which A1 and B1 displayed a large electrophoretic difference on both native and SDS-PAGE gels. The TF cDNAs corresponding to variants A1 and B1 were cloned and sequenced from A1A1, A1B1 and B1B1 individuals, and their deduced amino acid sequences were analyzed. Substantial amino acid variation occurred between variants A1 and B1, with significant differences in peptide length, theoretical molecular weight (Mw) and isoelectric point (pI). No potential glycosylation sites were observed in the two amino acid sequences, which excluded the possibility that carbohydrate difference might cause electrophoretic variation among the TF variants. Further analysis suggested that the distinct electrophoretic mobility of the two variants A1 and B1 by SDS-PAGE resulted from their Mw difference, while the difference by the native PAGE could be explained by their pI variation. Furthermore, genomic DNA fragments containing the transferrin alleles were amplified and subjected to RFLP analysis in A1A1, A1B1 and B1B1 individuals. The data revealed characteristic banding patterns for each TF genotype, and demonstrated that the TF alleles A1 and B1 could be used as a co-dominant marker system. The initial work relating to the goldfish TF variants will benefit the understanding of the evolutionary and functional significance of TF polymorphism in fish.     

Electrophoretic Mobility of Amylase in Drosophilids Indicates Adaptation to Ecological Diversity

Understanding the significance of electrophoretic variation is of interest for both ecological and evolutionary genetics. Although there has been a very active neutralist–selectionist debate about the patterns of electrophoretic variation in natural populations, it is only recently that charged amino acids have been shown to be important in enzyme adaptation. In this study we carried out a broad electrophoretic survey of amylase variation in 150 species of Drosophilids. The distribution of amylase electromorphs was found to be correlated with the geographical origin of the flies. Generally the faster migrating variants are found in warmer temperatures. There is also a correlation with the feeding habits of the species, in particular, fungus feeders consistently showed a deviating pattern of electrophoretic mobility. These correlations between ecological diversity and electrophoretic patterns indicate that at least some of the changes in charged amino acids are adaptive, and result from selection to cope with specific environments.     

De Novo mutation-like events observed at the 6PGD locus of the Japanese quail,and the principle of polymorphism breeding more polymorphism

In our stock of Japanese quail, four alleles which specify electrophoretic variants A, B, C, and D of an enzyme, 6PGD, are maintained. Analysis of the progeny from a mating which should have produced only known types of heterozygotes enabled us to detect a great variety of mutation-like events which affected the germ cells of the parents. A total of 1011 progeny from 26 such matings were typed for their 6PGD phenotype. Eleven showed unexpected phenotypes, some of which were apparent products of deletions or duplications. Thus, it appeared that the spontaneous rate of occurrence of all the mutation-like events per 6PGD locus per generation approaches 1×10^–2 in Japanese quail. All 11 mutation-like events occurred in the heterozygous parents. Furthermore, 8 of the 11 parents were A/D heterozygotes. A and D show the greatest difference in their electrophoretic mobility, which suggests that two variant subunits differ by several amino acid substitutions rather than by a single amino acid substitution. Of the 11 unexpected progeny, three received new, hitherto nonexistent electrophoretic variants from one of the parents. Perhaps there is a principle that mutation-like events are more likely to occur in germ cells of the parent which is heterozygous for extremely different alleles. This would imply that the new electrophoretic variants presently observed were produced by intracistronic recombination.This work was supported in part by a grant (CA 05138) from the National Cancer Institute, U.S. Public Health Service.     

Three new phenotypes of human red cell acid phosphatase: ACP1FA,ACP1GA,and ACP1GB

Summary Three new phenotypes of human erythrocyte acid phosphatase (ACP₁) have been detected and found to be unique by direct comparison with previously identified ACP₁ variants. One of these new electrophoretic variants, labeled as ACP₁FA, has been detected in the Hispanic population of California. The electrophoretic variants identified as ACP₁GA and ACP₁GB have been detected in a black family in North Carolina. A family study has shown that ACP ₁ ^G is transmitted as an allele of ACP₁.