Immunological studies on erythrocyte phosphoglucomutase isozymes

Human erythrocytes (phenotype PGM1 a1 or PGM1 a3) contain two sets of phosphoglucomutase isozymes, produced by the expression of the PGM1 and and PGM2 loci. The two sets are constituted each by two forms, of which that called "secondary" is thought to derive from the post-translational modification of that called "primary". Cross-reactivities of these isozymes were studied by means of monospecific rabbit antibodies against purified human red cell PGM1 and PGM2 "primary" isozymes. The results show that the PGM1 and PGM2 forms are not immunologically related and provide further proof of the post-synthetic origin of "secondary" isozymes and of the multifunctionality of PGM2 phosphoglucomutases.     

Unusual electrophoretic patterns for phosphoglucomutase and fumarase in a population of Lecithochirium rufoviride (Trematoda: Hemiuridae), a parasite of Conger conger.

Electrophoretic analyses of phosphoglucomutase (PGM) and fumarase (FH) in a population of Lecithochirium rufoviride parasitizing Conger conger, revealed 2 independent activity zones for each enzyme on starch gel electrophoresis. However, some individuals exhibited only 1 activity zone for 1 or both enzymes. The banding patterns observed strongly suggest that (1) PGM is coded by 2 polymorphic loci, Pgm-1 (expressed in all individuals) with allelic frequencies not significantly different from those expected under Hardy-Weinberg equilibrium, and Pgm-2 (expressed in a subset of individuals); and (2) FH is coded by 2 loci, Fh-2 (monomorphic and expressed in all individuals) and Fh-1 (expressed in a subset of individuals). A high degree of concordance (88.75%) was observed between the expression and nonexpression of Pgm-2 and Fh-1. The most likely explanations for these findings are either variation in enzyme expression with developmental stage or the presence of null alleles at high frequencies in the population.     

同工酶差异位点分析在蔬菜杂交种纯度检测中的应用

用１０种同工酶和蛋白质和分析体系,分析了７种重要蔬菜的７１个杂交种与其亲本之间的差异位点以及这些差异位点用于杂交种纯度检测的可能性和存在的问题。试验表明,作物的不同种类,杂交种与亲本之间的亲缘关系以及作物种类的遗传多态性,都会影响同工酶差异位点产生的多寡,从而影响到这一技术是否能用于此种作物的纯度检测,分析了不同同工酶在不同蔬菜中的多态性以及在蔬菜种子纯度检测中的表现。     

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.     

Inheritance of trunk banding in the tetra (Gymnocorymbus ternetzi Characidae)

The tetra (Gymnocorymbus ternetzi) exhibits two phenotypes associated with trunk banding. Fish possess either a smoky-gray coloration with two prominent black vertical bands located directly behind the operculum (black tetra) or a lighter coloration and lack these bands (white skirt tetra). Segregation patterns observed from the progenies of 11 different crosses suggest that the inheritance of these phenotypes is controlled by two autosomal loci acting in a complementary fashion, with dominance at both loci required for the expression of the darker, banded phenotype.     

Isozyme electrophoresis patterns of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China

An enzyme analysis of the liver fluke, Clonorchis sinensis from Kimhae, Korea and from Shenyang, China was conducted using a horizontal starch gel electrophoresis in order to elucidate their genetic relationships. A total of eight enzymes was employed from two different kinds of buffer systems. Two loci from each enzyme of aconitase and esterase (alpha-Na and beta-Na); and only one locus each from six enzymes, glucose-6-phosphate dehydrogenase (G6PD), alpha-glycerophosphate dehydrogenase (GPD), 3-hydroxybutyrate dehydrogenase (HBDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), and phosphoglucomutase (PGM) were detected. Most of loci in two populations of C. sinensis showed homozygous monomorphic banding patterns and one of them, GPD was specific as genetic markers between two different populations. However, esterase (alpha-Na), GPD, HBDH and PGI loci showed polymorphic banding patterns. Two populations of C. sinensis were more closely clustered within the range of genetic identity value of 0.998-1.0. In summarizing the above results, two populations of C. sinensis employed in this study showed mostly monomorphic enzyme protein banding patterns, and genetic differences specific between two populations.     

Purification and partial characterization of the phosphoglucomutase isozymes from human placenta

We have developed a simple procedure for the purification of phosphoglucomutase (PGM) isozymes from human placenta of healthy women. The technique involves the ammonium sulfate fractionation, ion-exchange and dye-ligand chromatographies. By this method we obtained homogeneous isozyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci. The final specific activities were 1134.6-1441.8 units/mg for PGM1 forms and 40.2-46.5 units/mg for PGM2 forms. On SDS-polyacrylamide gel electrophoresis analysis, the final preparations gave a single protein band of 58,500 and 69,000 Mr for PGM1 and PGM2 isozymes, respectively. These forms have the same kinetic properties, but from the substrate specificity experiments we have found that PGM2 forms are more effective for catalyzing the phosphoribomutase and glucose 1,6-bisphosphate synthase reaction than PGM1 forms. All these properties are shared by the same isozymes previously isolated from human erythrocytes but in this procedure the use of human placenta for the PGM isozymes purification takes advantage of high specific activity of PGM in the extracts of this tissue as well as obtaining highly homogeneous protein suitable for studies at molecular level.     

Evolutionary genetics of Metridium senile. II. Geographic patterns of allozyme variation

Electrophoretic surveys have demonstrated that populations of the sea anemone Metridium senile along the northeast coast of the United States are polymorphic at four enzyme loci. Phosphoglucose isomerase (PGI) has two alleles in most populations, phosphoglucomutase (PGM) has three alleles, and two leucine aminopeptidase loci have two common alleles each. Phosphoglucose isomerase displays clinal variation and an apparent association with environmental temperature. Phosphoglucomutase shows clinal variation north of Cape Cod for two of the three alleles, while the two leucine aminopeptidase loci are not clinal. All loci show a great deal of variation in populations on Cape Cod, but there is no apparent systematic pattern to this variation. Temperature may be a selective agent in the maintenance of the PGI and PGM clines, although other possibilities cannot presently be completely excluded.Supported by Grant T-4 from the Health Research and Services Foundation, NSF DEB77-14442, NIH GM25809, and NIH GM28024.     

Gametic disequilibrium in populations of reindeer Chukchi and Asian Eskimos

Ten local populations of Reindeer Chukchi and four local populations of Asiatic Eskimos were surveyed for variation of nine polymorphic loci (MN, Ss, Cc, Ee, Duffy, AcP, PGM1, Hp, G3M). Pairwise linkage disequilibria were estimated from multilocus genotype frequencies using the Hill's method. Large amounts of linkage disequilibrium were found for the two pairs of linked genes MNSs and CcEe. The same pattern of non-random association between unlinked loci PGM1 and AcP was found in all subpopulations of both tribes that is induced by natural selection.     

Inheritance of muscle and liver types of supernatant NADP-dependent isocitrate dehydrogenase in rainbow trout (Salmo gairdneri)

The genetics of allelic variation for NADP-dependent isocitrate dehydrogenase (IDH-s) found in the supernatant of liver and white muscle extracts of rainbow trout (Salmo gairdneri) was examined. Twenty progeny from each of 50 controlled matings were examined for IDH phenotypes. Progeny data clearly indicated that the IDH-s variation in the muscle is controlled by two loci—one fixed and one with two alleles producing molecules of different electrophoretic mobilities. IDH-s variation in the liver is controlled by two disomic loci which code for four alleles. No linkage between the loci controlling IDH-s in the liver and the loci controlling it in the muscle was detected.     

Isozyme variation and the reproductive biology of Hydrocharis morsus-ranae L. (Hydrocharitaceae)

Electro-phoretically detectable isozyme variation was studied for 17 enzyme systems in several N American populations of the introduced aquatic plant Hydrocharis morsus-ranae and in two European populations. Twenty-nine loci were inferred from progeny, adult and turion enzyme banding patterns with 28 of these loci homozygous in all individuals studied. Malate dehydrogenase-1 (MDH-1) was the only locus which could be interpreted as multi-allelic and heterozygous. Twenty-seven of 76 seedlings assayed showed an age specific expression for an alcohol dehydrogenase locus (ADH-2) never seen in adults or turions. Since all adults sampled were phenotypically identical at all loci assayed, it is possible that only one isozyme genotype of this species is present in N America. European turion data further indicated that the populations studied were identical to N American plants sampled at all loci except EST. Therefore, although H. morsus-ranae is dioecious, outcrossing appears to involve substantial inbreeding. Connections between extensive inbreeding and the failure of effective sexual reproduction are considered.     

Comparative study of red cell enzymes in Mirpur (a Portuguese-descendant community) and a neighbouring Hindu population of Gopalchak

Five enzyme systems (ACP1, PGM1, MDH, LDH and EsD) have been studied in Mirpur and Gopalchak. The allele PGM13 is present in polymorphic frequency in Mirpur. In respect of ACP1 and PGM1 loci, these two populations differ significantly.     

Patterns and levels of genetic differentiation in North American populations of the Alaskan wheatgrass complex

Levels and distribution of genetic variation were assessed using six allozymes in 27 populations of Alaskan wheatgrass (Elymus alaskanus) from different locations in Canada, USA, Greenland and Russia to obtain information on the genetic structure of these populations. The enzyme systems were ACO, DIA, GPI, MDH, PGM and SKD. Allozyme variation at the species level was high, with 64.3% (Ps) of the loci being polymorphic, an average number of alleles per locus of 1.9 (As), and an average genetic diversity of 0.17 (Hes). Differentiation was found in the populations studied, with the following findings: (1) statistically significant differences were found in allele frequencies among populations for every polymorphic locus (P < 0.001); (2) 63% of the total allozyme variation at polymorphic loci was partitioned among populations (GST = 0.63); (3) relatively low mean genetic distances between the populations were obtained (mean D = 0.029); (4) the genetic structure of Russian populations are clearly distinct from the other populations, the cluster and principal component analyses revealed the same genetic patterns of relationships among populations. This study also indicates that E. alaskanus contains different levels of allozyme variation in its populations. Furthermore, some banding patterns at the loci Aco-1, Aco-2, Gpi-2, Mdh-1, Skd-1, Skd-2 can be used as markers to identify individual populations.     

Allozyme frequencies in western European populations of Asellus aquaticus (L.) Isopoda and their association with water pollution

A study was made of allozyme variation in western European populations of Asellus aquaticus. Two enzymes were examined glucose phosphate isomerase (CPI) and phosphoglucomutase (PGM).
The populations show significant latitudinal clincs for GPI and PGM with the GPI-2 and PGM-2 alleles increasing in more southerly latitudes. While GPI-2 frequencies in British populations are consistent with the overall clinc, considered separately they show a reverse cline. This appears to be the result of a strong association of GPI-2 frequencies with water pollution combined with the geographic distribution of the polluted sites.
The association of high GPI-2 frequencies with high levels of water pollution is present in two independent sets of populations in Britain, and is more specifically associated with low levels of environmental oxygen. This latter association also occurs independently in temporal studies made at three locations. However, an additional temporal association of high GPI-2 frequencies with high temperatures also exists, consistent with the observed latitudinal cline. A study of adult mortality by oxygen deprivation at different temperatures suggests that both temperature and oxygen concentrations may act selectively with the GPI 2-2 genotype to be favoured under certain conditions. In contrast to GPI, PGM allozymes do not appear to be associated with pollution related variables.
The findings of the study point to selection acting on the two loci examined, though most convincingly for GPI. However, it remains to be demonstrated that selection is acting directly on the loci, and if so, how it operates to produce the patterns of allozyme variation observed in nature.     

Analysis of genetic variation within clonal lineages of grape phylloxera (Daktulosphaira vitifoliae Fitch) using AFLP fingerprinting and DNA sequencing.

Two AFLP fingerprinting methods were employed to estimate the potential of AFLP fingerprints for the detection of genetic diversity within single founder lineages of grape phylloxera (Daktulosphaira vitifoliae Fitch). Eight clonal lineages, reared under controlled conditions in a greenhouse and reproducing asexually throughout a minimum of 15 generations, were monitored and mutations were scored as polymorphisms between the founder individual and individuals of succeeding generations. Genetic variation was detected within all lineages, from early generations on. Six to 15 polymorphic loci (from a total of 141 loci) were detected within the lineages, making up 4.3% of the total amount of genetic variation. The presence of contaminating extra-genomic sequences (e.g., viral material, bacteria, or ingested chloroplast DNA) was excluded as a source of intraclonal variation. Sequencing of 37 selected polymorphic bands confirmed their origin in mostly noncoding regions of the grape phylloxera genome. AFLP techniques were revealed to be powerful for the identification of reproducible banding patterns within clonal lineages.     

Different karyotype patterns among allopatric Pinus nigra (Pinaceae) populations revealed by molecular cytogenetics

To examine variation and taxonomic recognition of Pinus nigra (European black pine) at the intraspecific level, chromosomal distribution of 5S and 18S-5.8S-26S rDNA loci revealed by fluorescent in situ hybridisation (FISH) and fluorochrome banding with chromomycin A(3) and DAPI were analysed among allopatric populations belonging to different subspecies. Despite prevalent opinion on predominantly conserved and homogenous conifer karyotypes, several patterns were observed. Surprisingly, interstitial 18S rDNA loci and DAPI heterochromatin staining after FISH showed variations in distribution and localisation. Three subspecies shared a pattern with nine 18S rDNA loci (ssp. nigra, pallasiana and laricio) while ssp. dalmatica and salzmannii had eight rDNA loci. DAPI banding displayed two patterns, one with a high number of signals (ssp. nigra, pallasiana and dalmatica) and the other with a lower number of signals (ssp. salzmannii and laricio). We conclude that our results cannot provide proof for either classification scheme for the P. nigra complex, but rather demonstrate the variability of different heterochromatin fractions at the intraspecific level.     

Isozyme Variation in a Tropical Pioneer Tree Species (Cecropia obtusifolia,Moraceae) with High Contents of Secondary Compounds1

We define 48 allozyme loci for a tropical pioneer tree species, Cecropia obtusifolia Bertol, which has high contents of secondary compounds. Our goals were to find the effects of extraction procedures on artifacts and variation in resolution of enzyme banding patterns; to explore the relationship among the variation of the loci sampled and the enzymes' molecular structure, metabolic function and substrate; to obtain estimates of the genetic variation in this species at Los Tuxtlas rain forest (México) and to explore the variation of allelic frequencies in six successive life-history stages of the species. The resolution of the isozymes bands and the actual banding pattern varied with the type and age of tissue, the collection and storage procedures, the extraction buffer, and other loading and running procedural details. However, artifactual variation was eliminated with a new extraction buffer for species with high contents of secondary compounds. Of the 26 enzymes resolved for C. obtusifolia, we found that enzymes with a greater number of substrates and an oligomeric quaternary structure tended to be more variable than their counterparts, but the relationship was not statistically significant. The proportion of polymorphic systems varied significantly with the metabolic pathway and the function of the enzymes. Enzymes involved in starch synthesis are significantly more variable (p < 0.05) than all others, except those involved in amino acids metabolism and the proportion of polymorphic enzymes is also significantly associated with the hnction of the enzyme, the hydrolases and isomerases are significantly more variable than lyases and oxidoreductases enzymes. The percentage of polymorphic loci for C. obtusifolia was estimated at P = 27.1%. The effective number of alleles was estimated at ne = 1.3 and ne = 2.4 for all loci and only polymorphic ones respectively and the average heterozygosity (H) for all 48 loci was estimated at H = 0.05. Allele frequencies varied throughout the life-cycle of the species, with significant differences for some alleles and loci among some life-cycle stages. “Tree seeds” allele frequencies differ significantly (P < 0.05) from “rainy dispersed seeds” in 7 of 8 loci and from “soil seeds” in Six of eight loci. Allele frequencies of all three seed categories (“tree seeds”, “rainy dispersed seeds”, and “soil seeds”) differed strongly from established individuals (seedlings, juveniles and adults), while allele frequencies of established individuals are relatively similar to one another. Seedling allele frequencies at most loci were also significantly different from those found in seeds collected from trees, seed-rain, and soil. Two alleles (at GOT-2 and FE-2) were only found in soil seeds and one allele (at LAP-2) was only found in seedlings.     

Diversity and inheritance of inter-simple sequence repeat polymorphisms in Douglas-fir (Pseudotsuga menziesii) and sugi (Cryptomeria japonica)

We studied inter-simple sequence repeat (ISSR) polymorphism and inheritance in Douglas-fir [Pseudotsuga menziesii (Mirb.) Franco] and sugi (Cryptomeria japonica D. Don) megagametophytes using primers that anneal to simple repeats of various lengths, sequences, and non-repetitive motifs at the 5 and 3 ends. Products were visualized on agarose gels with ethidium bromide staining. More than 60% of the 96 primers tested gave interpretable banding patterns in both Douglas-fir and sugi, and the useful primers were in complete agreement among species. Dinucleotide repeat primers were the majority of those tested, and gave all of the useful banding patterns. The 24 best primers were used for segregation studies, yielding a total of 77 loci distributed among two Douglas-fir families and one sugi family. Approximately 90% of the 24 primers showed polymorphism within at least one of the three families. The average number of variable loci per primer was 1.6. Primers based on (AG) _n repeats gave the largest number of polymorphic loci; 16 primer-family combinations yielded 24 segregating loci. However, primer based on (GT) _n repeats gave the most loci per primer studied (mean of 2.0). All markers displayed apparent dominance (band presence vs absence), and all but three segregation ratios (4%) fit Mendelian expectations: Because they employ longer primers than do RAPDs, have a high degree of polymorphism, conform well to Mendelian expectations, and do not require use of acrylamide gels for analysis, ISSRs may be useful markers for PCR-based genome maps and population studies of conifers.Paper 3082 of the Forest Research Laboratory, Oregon State University