Electrophoretic Variants Research Articles

Developmental change in activity of red cell porphobilinogen deaminase and its electrophoretic variant in the Japanese population.

The activity of porphobilinogen deaminase (PBGD), an enzyme whose partial deficiency is associated with acute intermittent porphyria (AIP), changes during development. Little is known about the postnatal change of PBGD activity and the prevalence of its electrophoretic variant in the Japanese population. The activity of PBGD was measured fluorometrically in 194 infants aged 0-12 months, while isoelectric focusing of PBGD was performed in 400 healthy Japanese adults aged 20-45 years and 30 children with various hematological disorders aged 1-15 years. The PBGD level was 1.9 times higher in the neonates than in the adults, decreased abruptly during the first month of life, and reached the adult level at the age of 9 months. None of the 400 healthy Japanese adults and the 30 children with hematological disorders showed any electrophoretic variant. These results suggest that there is no need to consider any polymorphism in the gene dose study of PBGD and that the biochemical screening of AIP is applicable to since the late infancy.

Animal breeding and conservation genetics.

Conservation genetics in an animal breeding context relates both to questions of preservation of rare and endangered breeds or populations, and to utilization with planned genetic change to improve viability, productivity, and efficiency of production. In the developed world, preservation is the primary issue, and various organizations exist which are committed to the preservation of rare and endangered breeds. In the developing world, breeds as such often are not defined or recognized, but many local populations exist that are adapted to and integrated into existing production systems. The genotypes of at least some of these populations could well also be crucial for future production systems, but many are threatened, primarily by crossbreeding with breeds introduced from the developed world. However, not all can be conserved, and priorities will have to be set for preservation, for development (breeding programs) and for evaluation for future programs. Some priorities will be set for pragmatic reasons, but the primary rational reason must be that a breed is in some way genetically unique, and makes a substantial contribution to the genetic diversity of the species. Thus, measures of genetic distance are essential to quantify the degree of genetic differentiation among populations, but such measures must be based on a large sample of loci. Although this has been emphasized many times, it still seems not to be adequately appreciated, and the effect of using a limited sample of loci is illustrated with an example from swamp buffalo populations. Comparative estimates of distances based on electrophoretic variation and direct DNA variation (both mitochondrial and genomic) are needed as a basis for future work on conservation of the global domestic animal diversity. Finally, studies of feral populations and wild relatives of domestic animals will provide a link between natural populations and domestic animal populations, and bring together these two areas, which to now have been largely separate.

Ethnic differences in enzyme and DNA polymorphisms of human tissue-specific alkaline phosphatases.

Enzyme and DNA polymorphisms and haplotypes of tissue-specific alkaline phosphatase genes were studied in Finns, Saamis and Swedes. In placental alkaline phosphatase (PLAP) restriction fragment length polymorphisms (RFLPs), found after digestion with RsaI and PstI, no significant population differences were observed. The PstI RFLP of the germ cell alkaline phosphatase (GCAP) locus showed significant allele frequency variations between Finns and Swedes (p = 0.014) and between Saamis and Swedes (p = 1 x 10(-4)). Electrophoretic enzyme variants of PLAP were studied in Finns and Swedes. The PLAP variant 18 (D in older nomenclature) was found at a polymorphic frequency in the Finnish sample. In all populations, strong linkage disequilibria were found between the RFLPs and between RFLPs and electrophoretic PLAP types. There was, however, one notable exception: between the RsaI RFLP of PLAP and the PstI RFLP of GCAP no linkage disequilibrium was found. Two new RFLPs were observed in the Finnish population sample, a RsaI mutant site in PLAP with a frequency of 0.02 and a KpnI mutant site outside and upstream of the PLAP gene with a frequency of 0.036. In accordance with findings in previous studies at the enzyme level, PLAP also appeared to be more polymorphic than GCAP and intestinal alkaline phosphatase at the DNA level.

Joint Segregation of Allozymes in Catfish Genetic Crosses: Designation ofIctalurus punctatusLinkage Group I

Polymorphic isozymes visualized by starch gel electrophoresis and histochemical staining were examined in parents and 1,000 progeny of 24 single-pair matings of channel catfish Ictalurus punctatus and three interspecific crosses of channel catfish × blue catfish I. furcatus. Polymorphic loci that could be scored from fin tissue included aconitate hydratase (mAH*), glucose-6-phosphate isomerase (GPI-B*), glutathione reductase (GR*), phosphoglucomutase (PGM*), and UDPglucose-hexose-1-phosphate uridylyltransferase (UGHUT-2*). Inheritance of observed electrophoretic variation was consistent with Mendelian segregation of codominant alleles in progeny selected randomly from the individual crosses. Although mAH*, GPI-B*, PGM*, and UGHUT-2* appeared to assort independently within the limits of the data, strong evidence for linkage of GR* and PGM* was obtained (19.7% recombination, P ≤ 0.001 for data combined from two informative pedigrees); this linkage was designated Ictalurus punctatus linkage group I. This study represented a first step in assembly of a catfish gene map large enough to provide genetic markers for each catfish chromosome to enable detection of specific loci associated with traits of economic importance by genetic linkage analyses. For the polymorphic loci studied here, no consistent association of genotype with average body weight was observed.

INDUCED PROTEIN AND ISOZYME VARIATION IN VIGNA RADIATA VAR. PS 16

Electrophoretic variation of protein profiles, esterase and amylase isozymes was investigated in 24 h germinated M3 seeds of Vigna radiata var. PS 16. The patterns of induced variations suggest that there is a differential gene expression as well as gene activation due to mutagenesis. The results suggest that mutagenesis could be used for locating genetic markers for the identification of mutants and for the construction of linkage groups and gene mapping.

Molecular and morphological evidence for hybridization between two ecologically distinct grasshoppers (Melanoplus sanguinipes and M. devastator) in California

This paper applies the biological species concept to two ecologically distinct species of grasshopper, Melanoplus sanguinipes and M. devastator, by testing for reproductive isolation in the field in California. Two independent techniques for assessing gene flow between species were employed. Firstly, we examined male genitalic morphology in populations from the foothills of the Sierra Nevada where the two species are parapatric. Two genetically based genitalic traits that differed between allopatric populations of each species formed a cline in this zone. Males captured in the field from the region of parapatry resembled offspring from interspecific laboratory hybridizations. Secondly, we surveyed electrophoretic variation of populations from across California and used F statistics to estimate levels of gene flow within and between species. Results from both morphology and F statistics suggested that these grasshoppers are not reproductively isolated in nature but that gene flow between species is reduced relative to within-species. These field results were consistent with a laboratory study that showed partial but not complete hybrid egg inviability. Because the hybrid zone is centred along an ecological transition, this system offers an opportunity to investigate whether adaptive changes across the zone contribute to reduced gene flow between species.

High levels of genetic subdivision of marine and estuarine populations of the estuarine catfishCnidoglanis macrocephalus (Plotosidae) in southwestern Australia

The cobblerCnidoglanis macrocephalus (Valenciennes) is an endemic marine and estuarine catfish from southern Australia. Conflicting views on the degree of isolation of the estuarine populations underscore general questions about genetic divergence in coastal species. Although estuaries are widely recognized as ecologically important, little work has been done on their role in favouring genetic divergence. In order to estimate the extent of genetic subdivision among nearshore marine and estuarine populations, electrophoretic variation of enzymes was examined in seven marine and six estuarine populations of cobbler from sites spanning 1500 km along the southwest Australian coastline. Among all populations, the mean standardized variance in allelic frequencies (FST) for six polymorphic loci was 0.277, a high value comparable to those of other shallow-water teleosts whose life-history characteristics and habitat preferences restrict their dispersal capability. The pattern of genetic identities between populations showed divergence between west and south coast sites. Within these regional groups, however, there was substantial heterogeneity, much of which was associated with estuaries. Among all six estuarine sites, the averageFST was 0.333, 40% higher than the value of 0.237 for the marine sites. Low estimates of the genetically effective number of migrants suggest population subdivision between marine and estuarine environments and between similar habitat types. This study indicates the importance of habitat in affecting the connectedness of populations, even in apparently open marine systems.

Genetic structure of the scald pathogen (Rhynchosporium secalis) in South East Australia: implications for control strategies

A survey of electrophoretic variation in 89 isolates of Rhynchosporium secalis collected from cultivated and wild barley grass in Victoria, New South Wales and Tasmania detected an average of 2.5 alleles at each of 11 loci in 5 enzyme systems. At five loci, two alleles each occurred at frequencies exceeding 0.2. Comparison of the frequency of alleles at these loci showed little association with host or geographic origin, pathogenicity of the isolates or with the allelic state at other loci. Given that this pathogen reproduces asexually only, this lack of association confirms previous suggestions that some means of asexual recombination is important in this pathogen. Furthermore, the results suggest that the most effective means whereby long-term control over R. secalis will be achieved is through the use of both pathotype-specific and field resistance complemented by other strategies like the use of fungicides and possibly varietal mixtures.

Genetic structures of the B2 and B1 Escherichia coli strains responsible for extra-intestinal infections

Escherichia coli strains causing human extra-intestinal infections may be divided into two groups, B1 and B2 according to the electrophoretic patterns of carboxylesterase B. This study compares the restriction fragment length polymorphism (RFLP) of ribosomal DNA (rDNA) for 45 B1 strains and 45 B2 strains to examine the genetic structure of B2 strains and to distinguish them from B1 strains. The isolates were chosen for diversity in their allozymes of esterases, B, A, C and I, their production of virulence factors (alpha-haemolysin, mannose resistant haemagglutinin and cytotoxic necrotizing factor) and certain O antigens, and their pathological and geographical origins. DNA was digested with HindIII and BamHI restriction enzymes and analysed by Southern blotting. The resulting rDNA RFLP patterns of B2 strains were distinct from those of the B1 strains. Moreover, the B2 strains appeared to be less heterogeneous than the B1 strains. The B2 strains gave 13 ribotypes (resulting from the combination of the rDNA RFLP patterns obtained with HindIII and BamHI digestions) while the B1 strains gave 32 ribotypes. Correspondence analysis of the data showed that several clusters of strains were identified in the B2 strains by particular ribotypes, certain associations of esterase B and A electrophoretic variants, O serotypes and virulence factor production. In contrast, these parameters appeared to be unrelated in the B1 strains, reflecting their heterogeneity. These findings, which differentiate two levels of genetic heterogeneity within E. coli pathogenic isolates, indicate that the B2 strains constitute a phylogenetically distinct group within the species.

Electrophoretic variation among the five polish populations of the bank vole

Electrophoretic variation in proteins encoded by 21 loci was analysed in five populations of the bank vole (Clethrionomys glareolus) from southern and eastern Poland. Intrapopulation variation is high (P=19.0–33.3%, H=0.039–0.067); the average Nei's distance among populations equals 0.193. Comparatively high FIS (0.306) values suggest high levels of differentiation. Variation in allele frequencies (FST is high (ranging from 0.137 to 0.572) and reflects considerable geographic genetic heterogeneity. Genetic and geographic distances are not consistently associated.

Origin of late lactation protein from beta-lactoglobulin in the tammar wallaby.

Two of the dominant whey proteins of the tammar wallaby, beta-lactoglobulin (BLG) and late lactation protein (LLP), have been positively identified on gradient polyacrylamide gradient gels by N-terminal protein sequencing. The origin of the electrophoretic variation present in these proteins has been found to be principally genetic. The variation was examined in 97 lactating female tammar wallabies, of which 10 were followed throughout lactation to determine if any developmental variation existed. The only distinguishable developmental difference results from the previously described appearance of LLP at the beginning of phase III of lactation (approximately 180 days after birth of the young). Electrophoretic analysis of BLG implies that it exists in monomeric, dimeric, and tetrameric forms. Very strong linkage disequilibrium, indicative of close linkage, was found between alleles at the Blg and Llp loci. Examination of their cDNA sequences suggests that LLP and BLG represent an ancient gene duplication. Both have conserved amino acid residues characteristic of the lipocalins. The results imply that the marsupial mode of lactation, and hence of reproduction, is also very ancient.

Biochemical and genetic studies on alkaline phosphatase ofCeratitis capitata

Two forms of alkaline phosphatase exist in the integument of the "white pupae" (wp) and dark pupae (dp) mutant strains of Ceratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the "dark pupae" mutant catalyze the hydrolysis of phosphotyrosine and beta-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited by L-tyrosine, but L-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome of Ceratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.

CDNA sequence of four purine nucleoside phosphorylase (Np) alleles in the mouse.

The molecular basis of four electrophoretic and activity variants of purine nucleoside phosphorylase in the mouse was examined by amplification and sequence analysis of cDNA. Compared with the cDNA coding sequence for C3H/HeHa designated Npa, there were five nucleotide changes for C57BL/6J, Npb; three for MOLF/Ei, Npc; and five for SPRET-1, Npd. There was only a single codon change between Npa and Npb, the deduced substitution of threonine 176 by serine. Similarly, there was only a single codon change between Npa and Npc, resulting in substitution of methionine 258 by lysine. There were three codon changes between Npa and Npd, resulting in substitution of glutamate 22 by lysine, threonine 39 by alanine, and aspartate 152 by glutamate. These amino acid substitutions-neutral to neutral, neutral to basic, and acidic to basic--are in agreement with the electrophoretic properties of the gene products of Npa relative to Npb, Npc, and Npd previously described by isoelectric focusing. Codon differences were confirmed by PCR-RFLP or single nucleotide primer extension analysis and extended to include the assignment of other strains as Npa: C3H/HeHa, DBA/2J, CLA, Posch-2; or Npb: C57BL/6J, C57L/J, C58/J. Both RFLP analysis of amplified genomic DNA and Southern analysis are consistent with single but unique Np alleles present in the C3H/HeHa and C57BL/6J genomes. As these data do not support the previous two-loci, Np-1 and Np-2, classification, we propose and employ a new single locus multiple allele classification for Np on the basis of the sequence analysis.

Expression of α-glycerophosphate dehydrogenase during the development and aging of malaria vector Anopheles stephensi (Diptera: Culicidae)

α-Glycerophosphate dehydrogenase (GPDH) expression during the complete development and aging of Anopheles stephensi has been studied by electrophoretic and spectrophotometric techniques. Single gene locus seems to govern the developmental expression of three isozymes numbered from GPDH-1 to GPDH-3 in order of decreasing mobility. GPDH-3 was present in all the stages of development. GPDH-1 disappeared during senescence. GPDH-2, the band of intermediate mobility, was adult specific. GPDH-1 has been indicated to be more suitable for energy metabolism and GPDH-3 for lipid biosynthesis. Three codominant alleles control the four electrophoretic variants revealed in the laboratory strain. Quantitatively, the activity of GPDH continuously increased during development and showed its peak on day-4 adults of both sexes. However, the activity decreased during senescence. Tissue and sex specific differences in isozymes pattern have also been analysed. Changes in isozymes and specific activity have been correlated with the various physiological and morphological events occurring during development.

ANNUAL DIFFERENCES IN FEMALE REPRODUCTIVE SUCCESS AFFECT SPATIAL AND COHORT-SPECIFIC GENOTYPIC HETEROGENEITY IN PAINTED TURTLES.

Long-term ecological data were used to evaluate the relative importance of movements, breeding structure, and reproductive ecological factors to the degree of spatial and age-specific variation in genetic characteristics of painted turtles (Chrysemys picta) on the E. S. George Reserve in southeastern Michigan. Estimates of the degree of spatial genetic structuring were based on the proportion of total genotypic variance partitioned within and between subpopulations (inferred from hierarchical F-statistics based on variation at 18 protein loci), and in terms of gene correlations (co-ancestry among individuals derived from reproductive data on full-sib families of females nesting at specific nesting areas). Little variation in allele frequency was observed among turtles from different marshes (Fmt = 0.003), though significant variation was observed among turtles from different nesting areas associated with each marsh (Fnm = 0.046). Gene correlations among individuals within nesting areas varied greatly over years (0.032-0.171; mean = 0.069) and were negatively correlated to the proportion of females that successfully nested during each year. General concordance between independent estimates of genotypic correlations (i.e., Fnm derived from protein electrophoretic variation vs. mean co-ancestry) suggests that allozyme data, when collected over spatial scales consistent with species behavioral characteristics and reproductive ecology, may accurately reflect the apportionment of gene diversity within and among subpopulations. The magnitude and patterning of allelic variation among nesting areas and individuals appears to be primarily a function of gametic correlations among members of full-sib families, irrespective of the degree of gene flow or female nesting-site fidelity. Comparisons of genetic characteristics among 11 cohorts (1974-1984) revealed that heterozygosity (H) and inbreeding coefficients (F) varied greatly. Cohort estimates of H and F were correlated to female nesting success and to estimates of co-ancestry for the same years. Results clearly reflect the concomitant importance of ecological factors (principally the proportion of the female population that successfully produce offspring during each year) in determining the magnitude and patterning of gene correlations within and among groups, and to the genotypic composition of offspring born during each year.

Ripening-Related Polygalacturonase cDNA from Avocado

During the ripening of avocado (Persea americana) fruit, extensive cell wall degradation leads to a dramatic softening of the mesocarp tissue. Two enzymes have been suggested to play a critica1 role in this ripening-associated cell wall degradation in many fruits, including avocado, viz. cellulase (endo-~-1,4-glucanase) and endo-PG (Awad and Young, 1979; Fischer and Bennett, 1991). Although the avocado cellulase cDNA (Tucker et al., 1987) and gene (Cass et al., 1990) have been cloned and sequenced, the avocado PG has not previously been characterized at the molecular level. The ripe avocado fruit is known to contain numerous electrophoretic variants of the PG protein (Kanellis et al., 1991), but whether these represent different gene products or posttranslational modifications of one or more primary polypeptides is unknown. In tomato fruit, the various PG isoforms that accumulate during ripening (PG1, PG2A, and PG2B) are derived from a single gene (Fischer and Bennett, 1991). Using heterologous hybridization with a tomato cDNA probe, we have identified a putative avocado PG cDNA (pAVOpg) from a library generated with RNA from ripe avocado fruit (Table I). The cDNA contains an insert of 1725 bp that detects a 1900-nucleotide mRNA on hybridization to ripe fruit RNA and no detectable signal in mRNA from unripe fruit. This pattem of expression during ripening in avocado parallels that previously reported for PG enzyme activity (Awad and Young, 1979). Sequence analysis of the pAVOpg cDNA revealed an open reading frame of 453 amino acids, which is similar in size to that reported for avocado PG protein (Kanellis et al., 1991). This putative avocado PG sequence shows close similarity to PG sequences derived from both tomato and com, 52 and 36% identity, respectively. In addition, it contains an octapeptide motif that is conserved in PG sequences derived from plant, fungal, and bacterial species (Bairoch, 1992). This octapeptide is contained within a span of 14 residues (TCGPGHGISIGSLG) that is identical among the known plant PG sequences and thus should allow characterization of other plant PG genes using degenerate oligonucleotide primers in polymerase chain reaction experiments.

Genetic consequences of an invasion through a patchy environment — the cynipid gallwasp Andricus quercuscalicis (Hymenoptera: Cynipidae)

AbstractOver the last 300–400 years, the cynipid gallwasp Andricus quercuscalicis has invaded northern and western Europe following human introduction of an obligate host plant, the Turkey oak (Quercus cerris) from south‐eastern Europe. In the introduced range, distances between Turkey oak patches are greater and the numbers of oaks in each patch are far lower than in its native range. These changes in spatial distribution of Turkey oak are predicted to result in high genetic subdivision of A. quercuscalicis in invaded areas relative to its native range. Allozyme electrophoresis was used to examine genetic variation in 823 gall wasps from 39 populations of A. quercuscalicis. No new electrophoretic variants were found in the invaded range and both allelic diversity and mean heterozygosity decreased significantly with distance from the native range. Spatial autocorrelation analysis and values of Wright's Fst indicate that differences in allele frequences between populations were substantially greater in the invaded range than in the native range. Spatial autocorrelation analysis also suggests that changes in allele frequencies across Europe are unlikely to be the results of selection, but rather of strong directional migration followed by limited gene flow between populations. Patterns of genetic differentiation across Europe suggest that populations of A. quercuscalicis have been founded sequentially from the east through a process of random genetic subsampling and not by source‐sink colonization directly from the native range.

Isolation and characterization of carboxylesterase E3 from Salmonella enteriea

Three esterases (Est-) hydrolysing alpha-naphthyl acetate: Est-E1, Est-E3 and Est-E4 produced by Salmonella enterica serovar Typhimurium, strain LT2 were separated by DEAE chromatography and gel filtration. Est-E3, the major component of this set of enzymes, clearly differed from the two other esterases by its apparent molecular weight, titration curve, substrate specificity and inactivation. Immunoglobulins raised against Est-E3 completely neutralized the activity of Est-E3 but did not react with Est-E1 or Est-E4; it showed no cross reaction with carboxylesterase B of Escherichia coli or with carboxylesterases from other enterobacteria. Est-E3 showed electrophoretic variants which were biochemically and immunologically detected in the seven subspecies of the genus Salmonella. These findings suggest that variants of Est-E3 are the products of very closely related loci originating from a common ancestral gene. The esterase could be a phylogenetic marker of the genus and a suitable molecular tool for taxonomy and epidemiology.

No association found between C3 alleles and scar hypertrophy

Serum C3 was examined for structural variants using high-voltage agarose gel electrophoresis at both pH 6.2 and 8.4, thermal stability and stability to attack by hydroxyl radicals, in 450 Chinese people in Hong Kong. The C3 allele frequencies for electrophoretic variants were found to be 0.9911 for C3 ∗S and 0.0089 for all other rare alleles. No variants were found in terms of stability at 54°C in the absence of Ca 2+ or at 62°C in the presence of Ca 2+, and in terms of stability to attack by hydroxyl radicals. Based on a recently proposed single gene hypothesis for scar hypertrophy, statistical analyses were carried out to compare the relevant frequency data of scar hypertrophy with C3 phenotype frequencies, both for the local Chinese population and a European Caucasian population. There is no evidence for any association between C3 alleles or phenotypes and the formation of hypertrophie scars.

Limited tryptic digestion near the amino terminus of bovine liver rhodanese produces active electrophoretic variants with altered refolding

When the enzyme rhodanese was partially digested by immobilized trypsin, it retained greater than 50% of its original activity although less than 10% of the undigested enzyme remained. The predominant daughter species were two 31-kDa polypeptides whose amino termini corresponded to either residue 44 or 45 of the enzyme's sequence. Following digestion, charged species were isolated by ion exchange chromatography. Denaturing electrophoresis revealed that a 4-kDa peptide remained associated with the 31-kDa fragment. This 4-kDa peptide appears to correspond to the amino-terminal 45 residues of rhodanese. Further proteolysis gave a 2.5-kDa peptide that dissociated under non-denaturing conditions without apparent change in migration of the 31-kDa fragment on SDS gels. Refolding of undigested, urea-denatured rhodanese restored much of its activity. Similar treatment of rhodanese following limited tryptic digestion resulted in no regain of activity. Refolding of a mixture of intact and digested rhodanese resulted in regain of activity appropriate for the amount of intact rhodanese in the sample, indicating that clipped rhodanese does not inhibit refolding of intact rhodanese. It is concluded that portions of the amino terminus of rhodanese are important in the enzyme's folding, but are not essential for the enzyme's sulfurtransferase activity.