Cytogenetic localization by variation in electrophoretic allozyme phenotype: Drosophila Odh

A gene-dosage effect is characteristic of eukaryotic structural genes and is therefore useful in gene mapping. However, attributing quantitative variations in enzyme activity to a gene-dosage effect or other putative regulatory loci can be suspect when the locus in question may be inducible by variations in culture conditions. The problem of controlling for allele-specific variations in activity and regulation can be circumvented in Drosophila melanogaster by the use of synthetic duplications and deficiencies in conjunction with enzyme polymorphism. A method for constructing segmental aneupliods heterozygous for electrophoretic variants of octanol dehydrogenase (Odh) is presented which permitted variations in allozyme phenotype and enzyme activity—which show a strict dosage dependency—to be produced simultaneously. The structural gene region for Odh was identified using T(Y;A) stocks and the deficiency M(3)S31 was used to assign the locus to polytene band region 86D1–4. With this method a segmental aneuploid survey of Drosophila for purposes of gene localization can be accomplished in one generation with appropriate stocks.This work was supported by National Institutes of Health Research Grant GM18678 awarded to E. Novitski.     

Biochemical polymorphism in the rat: Genetics of three electrophoretic variants and characterization of inbred strains

Nine inbred strains of the rat (Rattus norvegicus) were screened for differences in electrophoretically detectable proteins. Interstrain variation was observed for 7 of 26 proteins. Three of these variants have not been described previously: leucine aminopeptidase (Lap-1), major urinary protein (Mup-1), and seminal vesicle protein (Svp-2). Genetic analysis revealed two autosomal alleles for each of these polymorphisms. The loci Lap-1, Mup-1, and Svp-2 are linked neither to one another nor to the previously described Svp-1 and Es-4 loci. Each of the nine strains can be identified now by a specific set of monogenic markers.     

Allozymes characterize sibling species of bipolar Priapulida (Priapulus,Priapulopsis)

Electrophoretic polymorphism of glucosephosphate isomerase (Gpi^*) and phosphoglucomutase (Pgm ^*) polymorphisms were assayed in the bipolardisjunct species pairsPriapulus caudatus/P. tuberculatospinosus andPriapulopsis bicaudatus/Priapulopsis australis (phylum Priapulida). Numbers of genotypes fromGpi ^* alleles and 14Pgm ^* alleles generally did not show deviations from Hardy-Weinberg expectations in composite populations sampled over geographic distances up to 500 km linearly measured. This genetic pattern suggests efficient population cohesion in a phylum where pelagic larvae have not been observed. Sibling species in generaPriapulus orPriapulopsis from northern and southern polar seas did not share identical allozyme alleles.     

Latitudinal variation in octanol dehydrogenase and acid phosphatase allele frequencies in Drosophila melanogaster

Summary The octanol dehydrogenase (Odh) and acid phosphatase (Acph) loci of Drosophila melanogaster are each polymorphic for two electrophoretically detectable alleles. The frequencies of the Odh and Acph alleles have been analysed in populations sampled from up to a 40 ° latitudinal range in each of Australasia, North America and Europe/Asia. Odh ^S frequency is found to be significantly negatively associated with distance from the equator in all three zones. The relationship of Acph ^S frequency to distance from the equator is significant and negative in Australasia but neither significant nor consistent in sign in North America and Europe/Asia.     

Genetic features of root fungus-resistant scotch pine trees in artificial stands in the steppe zone of Ukraine

A comparative analysis of the allozyme variation in 18 loci of three steppe natural populations of Pinus sylvestris L. and a sample of 36 resistant trees from artificial stands at locations affected by root fungus (Heterobasidion annosum (Fr.) Bref.) is conducted. The resistant trees are characterized by lowest percentages of polymorphic allozyme loci, a fewer number of alleles, and number of genotypes close to the mean-population number, as well as a level of observed and expected heterogeny. In terms of composition and frequencies of multi-loci genotypes (Dia-1, Lap-1, Acp) that are responsible for the greatest contribution to the partitioning of the stands studied, the sample of resistant trees differs markedly from the natural populations.     

Development of microsatellite markers for the endangered freshwater pearl mussel Margaritifera margaritifera L. (Bivalvia: Unionoidea)

Freshwater pearl mussels (Margaritifera margaritifera L.) are among the most critically endangered freshwater invertebrates. We describe the isolation and characterization of the first microsatellite markers for this species, which were obtained by screening 4900 recombinant clones from two genomic libraries. Thirteen loci revealed polymorphisms as demonstrated on 42 tested individuals from four river drainages. Allelic richness ranged from two to 12 alleles and averaged 6.8 alleles per locus with heterozygosity levels varying from 0 to 0.850 for observed heterozygosity (H_O) and from 0.174 to 0.850 for expected heterozygosity (H_E). Deficiency of heterozygous genotypes was observed in eight of 13 loci.     

Genic heterozygosity, chromosomal interchanges and fitness in rye: any relationship?

Relationship between heterozygosity at allozyme loci, chromosomal interchanges and fitness was analyzed in a rye cultivar showing a polymorphism for such rearrangements. Nine allozyme systems (ACO, ACPH, GOT, GPI, LAP, MDH, PER, PGD and PGM) and five components of fitness (number of fertile tillers, total offspring, egg cell fertility, flowers/ear and seeds/ear) were studied. The estimated selection coefficients against interchange heterozygotes ranged from s = 0.12 to s = 0.34. A significant effect of the genic heterozygosity on some fitness components was observed in interchange heterozygotes (tillering and total offspring), in their standard homozygous sibs (flowers/ear and seeds/ear) and in the descendants of the crosses between standard karyotypes (flowers/ear, seeds/ear and egg cell fertility). However, the main effect was linked to genetic background associated to different crosses. Significant differences for Acph-1, Gpi-1, Lap-1, Mdh-1, Mdh-4, Pgd-2 and Pgm-1 loci were also found in some of these crosses although these differences were inconsistent. This suggests that probably the allozyme loci analyzed were not directly contributing to the fitness and that they are linked, in some cases, to different deleterious alleles depending on both cross and locus. This fact could support the local effect hypothesis as explanation although we do not discard the existence of some inbreeding level (general effect hypothesis) since all crosses and loci studied show a overall consistent trend of increased fitness with increased heterozygosity.     

Geographic pattern of allozyme and inversion polymorphism on chromosome O of Drosophila subobscura and its evolutionary origin

The chromosome O of Drosophila subobscura was studied with respect to genetic variability at three enzyme loci (Odh, Me, and Lap-4) and with respect to inversion polymorphism. Population samples were taken from seven localities along a north-south gradient from Sweden and Scotland to Tunisia.The chromosomal analysis revealed clinal frequency changes for gene arrangements from north to south. With the enzyme loci Odh and Me allele frequencies are similar throughout the distribution range. Both loci are located outside the common inversion complex O₃₊₄. On the other hand, frequency changes parallel to those of the gene arrangements were observed for the alleles of the Lap-locus. Nonrandom associations between Lap-alleles and the superimposed gene arrangements O_ST, O₃₊₄, O₃₊₄₊₈, and O₃₊₄₊₂₃ were found. These gene arrangements differ from each other with respect to allele frequencies at the Lap-locus but for a given gene arrangement the relative frequencies of Lap-alleles remain relatively constant along the north-south gradient. Thus allele frequencies at the Lap-locus can be predicted from inversion frequencies.These observations can be interpreted in such a way that the pattern of allozyme variation within gene arrangements is due to founder effects caused by the unique origin of inversions. The gene blocks in the different inversions seem to represent more or less separated gene pools. In polymorphic populations the coexistence of genetically differentiated inversions presumably gives rise to heterotic interaction.     

Fitness differences associated with Pgi SNP genotypes in the Glanville fritillary butterfly (Melitaea cinxia)

Allozyme variation at the phosphoglucose isomerase (PGI) locus in the Glanville fritillary butterfly (Melitaea cinxia) is associated with variation in flight metabolic rate, dispersal rate, fecundity and local population growth rate. To map allozyme to DNA variation and to survey putative functional variation in genomic DNA, we cloned the coding sequence of Pgi and identified nonsynonymous variable sites that determine the most common allozyme alleles. We show that these single‐nucleotide polymorphisms (SNPs) exhibit significant excess of heterozygotes in field‐collected population samples as well as in laboratory crosses. This is in contrast to previous results for the same species in which other allozymes and SNPs were in Hardy–Weinberg equilibrium or exhibited an excess of homozygotes. Our results suggest that viability selection favours Pgi heterozygotes. Although this is consistent with direct overdominance at Pgi, we cannot exclude the possibility that heterozygote advantage is caused by the presence of one or more deleterious alleles at linked loci.     

A long-term study on interactions between the Adh and αGpdh allozyme polymorphisms and the chromosomal inversion In(2L)t in a seminatural population of D. melanogaster

The Adh and αGpdh allozyme loci (both located on the second chromosome) showed considerable fluctuations in allele frequencies in a seminatural population of Drosophila melanogaster during 1972–97. Both long-term and short-term fluctuations were observed. The short-term fluctuations occurred within almost all years and comparison of allele frequencies between winters and summers showed significantly higher Adh^S (P < 0.001) and αGpdh^F (P < 0.01) allele frequencies in summers. Frequencies of these alleles were significantly positively correlated with environmental temperature, suggesting the adaptive significance of these allozyme polymorphisms. Frequency changes of the Odh locus (located on the third chromosome) showed no seasonal pattern and were not correlated with environmental temperature. Almost all short-term and long-term increases in Adh^S frequency were accompanied by a corresponding decrease in αGpdh^S frequency (r = –0.82, P < 0.001) and vice versa. Further analysis showed that gametic disequilibria between the Adh and αGpdh loci, which frequently occurred, were due to the presence of inversion In(2L)t located on the same chromosome arm and In(2L)t frequencies were positively correlated with environmental temperature. Gametic disequilibria between Adh and Odh and between Odh and αGpdh were hardly observed. Because In(2L)t is exclusively associated with the Adh^S/αGpdh^F allele combination, the observed correlated response in Adh/αGpdh allele frequencies is (at least partly) explained by hitchhiking effects with In(2L)t. This means that the adaptive value of the allozyme polymorphisms has been overestimated by ignoring In(2L)t polymorphism. Fluctuations in Adh allele frequencies are fully explained by selection on In(2L)t polymorphism, whereas we have shown that αGpdh frequency fluctuations are only partly explained by chromosomal hitchhiking, indicating the presence of selective differences among αGpdh genotypes in relation with temperature and independent of In(2L)t. Frequency fluctuations of αGpdh and In(2L)t are consistent with their latitudinal distributions, assuming that temperature is the main environmental factor varying with latitude that causes directly or indirectly these frequency distributions. However, the results of the tropical greenhouse population show no correlation of Adh (independent of In(2L)t) and Odh allele frequencies with environmental temperature, which may indicate that the latitudinal distribution in allele frequencies for these loci is not the result of selection on the F/S polymorphism in a direct way.     

Predatory blue crabs induce byssal thread production in hooked mussels

Abstract. Blue crabs (Callinectes sapidus) prey on hooked mussels (Ischadium recurvum) growing epizoically on oyster clumps in estuaries along the Louisiana coast. In prey size‐selection experiments, blue crabs preferred small mussels (<30‐mm shell length) to larger mussels, possibly because handling time increased with mussel size. When crabs were given a choice of solitary mussels versus mussels in clumps on oysters in the laboratory, mortality was lower by 86% in clumped mussels. However, no size selection by crabs occurred with mussels in clumps, likely because smaller mussels escaped predation in crevices between larger mussels or oysters. When individuals of two size classes of mussels were exposed to water containing the scent of crabs and of mussels consumed by blue crabs, an increase in byssal thread production was induced in all mussels, but byssal thread production rate was higher for small mussels than for large mussels. We conclude that increased predation risk for small mussels has resulted in higher size‐specific production of byssal threads, and that predator‐induced production of byssal threads, which may increase clumping behavior, may reduce their risk of mortality to predatory blue crabs.     

Heterosis in the flour moth,Ephestia kühniella

Four lines of Ephestia kühniella each homozygous for one of three different allozyme alleles at the Est-2 locus and two alleles at the Adh locus were crossed in order to study the extent of somatic, reproductive and adaptive heterosis in F₁ hybrids in comparison with the mean performance of the simultaneously reared inbred parent lines. With regard to adult weight and wing length (somatic heterosis) hybrids exhibit maximally 20% (males 10%) and 9% heterosis, respectively. As concerns the production of eggs and hatched larvae (reproductive heterosis) hybrids exceed the parental mean by 90% and 200%. Adaptive heterosis is realized by a shorter development period of the hybrids (maximally by 30%) as well as by significant lower variance of all metrical characters studied. In the F₂ the degree of heterosis diminishes. There is neither an excess of heterozygotes among the segregating allozyme genotypes nor superior performance of the heterozygotes concerning any one of the traits studied. Therefore, it is concluded that the pronounced heterosis in F₁ is not a single-gene-heterosis operating at the Est-2 and the Adh-locus.     

Temporal and Spatial Allozyme Variation in the South American Cactophilic Drosophila antonietae (Diptera; Drosophilidae)

Drosophila antonietae is an endemic South American cactophilic species that uses Cereus hildmaniannus rotting cladodes as breeding sites. We assessed temporal and spatial intrapopulational allozyme variation of two natural populations. Our results suggest that environmental variation (rain precipitation) is probably influencing allozyme temporal variation. Moreover, it seems that D. antonietae does not have intrapopulation structure and has N _ev (variance effective size) 83 and N _ec (number of adult flies that colonize each rotting cladode) = 21. The deficiency of heterozygotes found must be due to null alleles, a temporal Wahlund effect, or selection against heterozygotes. Assortative mating and inbreeding are discarded. This is the first report on allozyme variation in D. antonietae. It gives some insight on intrapopulational genetics through space and time for this species. This is important to understand its general genetic variability and will be essential to future works on the natural history and evolution of this species.     

Allozyme Loci Selection in the Pacific Mussel Mytilus trossulus under Stressful Conditions

The allozyme variability for 15 loci in two samples of the Pacific mussel Mytilus trossulus collected from a single giant cluster was investigated using the method of gel electrophoresis. One sample was subjected to short-term anaerobic stress and then to a longer aerobic stress, leading to the death of about 85% of the individuals. At some of the loci, significant differences in the genotypic and allele frequencies were found between the samples. The results are suggestive of the differential survival of mussels with different genotypes and alleles at some of the surveyed loci under stress. Our data are in agreement with the hypothesis of the adaptive significance of allozyme polymorphism.     

Purification and characterization of two allozymic forms of octopine dehydrogenase from California populations ofMetridium senile

Summary In northern and southern California populations of the plumose sea anemone,Metridium senile, octopine dehydrogenase occurs in two allozymic forms and these forms are distributed in a highly population-specific manner; the frequency of the slow allele (ODH ¹⁰⁰) is 0.875 in the northern (Bodega Bay) population while the frequency of the fast allele (ODH ¹⁰³) in the southern population (Santa Barbara) is 0.125. Purification techniques resulted in an increase in purity of approximately 400 fold. The enzyme is a monomer ofM _r 35,000 to 40,000. Though there is some flexibility in the amino acid substrate which the enzyme uses (arginine and lysine react similarly), the specificity for the keto acid is limited to pyruvate.The kinetic characters of the two allozymes ofMetridium senile ODH are very different with respect to type of substrate saturation (Fig. 4 and 5) and apparent Michaelis constants (K _m) for pyruvate, lysine, arginine, and octopine (Table 5), product inhibition by octopine (Fig. 2), and optimal activity with respect to pH (Fig. 3). The properties of the slow and fast allozymes resemble the kinetic properties of cephalopod brain and muscle tissue-specific isozymes (Table 7). The kinetic data indicate that the slow allozyme would not allow a great deal of accumulation of octopine in vivo, while the fast allozyme is poised markedly towards octopine production.When the data presented in this study are compared to various physiological findings of other investigators, it becomes evident that the probable in vivo function of ODH in sea anemones is to act, in a manner analogous to vertebrate LDH, during the short-term anaerobiosis associated with muscle contraction and locomotion. The population-specific distribution and the different functional properties of the two ODH allozymes are most likely related to the different degree of tidal exposure which the two populations experience in nature. Only the slow allozyme possesses the regulatory properties which would allow a shift to the alternative anaerobic pathways utilized during these longer exposure periods.Abbreviations ODH octopine dehydrogenase - LDH lactate dehydrogenase - PHI phosphohexose isomerase     

Genetic variation of an intestinal leucine arylaminopeptidase (Lap-1) in the mouse and its location on chromosome 9

Electrophoretic variation for an intestinal enzyme that cleaves L-leucyl-beta-naphthylamide has been discovered among inbred mouse strains. Several strains including related strains C57BL/6J, C57BL/10J, C57BR/cdJ, C57L/J, and C58/J demonstrate an electrophoretic band of this enzyme that is absent in other strains and stocks thus far observed. The enzyme is tentatively being called leucine arylaminopeptidase (LAP) and the variant genetic locus Lap-1. The presence of the band is determined by an allele designated Lap-1a. Homozygotes for the alternate allele, Lap-1b, are without the band and heterozygotes are, under our electrophoretic conditions, indistinguishable from Lap-1a homozygotes. Data from recombinant inbred lines and a B6D2F1 X DBA/2J backcross established linkage of Lap-1 to dilute (d) and supernatant malic enzyme (Mod-1) on chromosome 9 in the following order: Lap-1-d-Mod-1. The Lap-1 to d map distance was estimated to be 21.3 +/- 4.6 cM from backcross data and 8.1 +/- 4.8 cM from recombinant inbred lines.     

A comparative study of embryonic gene expression inDrosophila andEphestia

Summary The ontogeny of allozyme patterns has been studied in embryos ofDrosophilamelanogaster, which are doubly heterozygous for alleles specifying the slow and fast forms of alcohol dehydrogenase (ADH) and -glycerophosphate dehydrogenase (GPDH). The ontogeny of esterase-2 was studied in embryos and young larvae of the flour mothEphestia kühniella, which are heterozygous for two of the three existing esterase-2 alleles. In freshly laidDrosophila eggs only the maternal enzyme forms are present and during the first 15 hours of development the staining of these forms becomes progressively fainter. After 16 and 17 h, the paternal and hybrid bands of ADH and GPDH respectively become obvious. Before hatching, the intensity distribution in the three-banded pattern of reciprocal hybrids is asymmetric in favour of the persisting maternal enzyme form. InEphestia embryos, however, there is no persistence of the maternal esterases. In all reciprocal heterozygotes a three-banded pattern suddenly appears 96 h after egg deposition, indicating synchronous activation of both parental alleles. The relative intensity distribution in the hybrid patterns approaches that of the mature larvae stepwise and in an allele-specific manner. This result and the fact that the various heterozygous types exhibit unequal total activities suggest that the Esterase-2 alleles have different activities, which are fixed late in embryogenesis.     

Confirmation of intraspecific crossing and single and joint segregation of biochemical loci ofVolvariella volvacea

Nineteen lines ofV. volvacea were obtained from worldwide sources and analyzed for allozyme activity. Twelve loci (Aat, Acp-2, Dia, Est-4, Gipi, Lap-1, Pep-GL, Pep-LLL-1, Pep-LLL-2, Pgm, Pgd, andSod) were monomorphic; 5 loci (Ada, Gpt, Mpi, Np, andPep-PAP) were polymorphic. Nine unique genotypes were distinguished among the 19 lines. Multilocus enzyme electrophoresis was used to confirm intraspecific crosses between putative homokaryons. Putative homokaryons were recognized by electrophoresis of single-spore-derived cultures and used as breeding stock. These breeding stocks were grown together in dual culture on agar, and selections were made from the interaction zone. These selections were then transferred to make spawn. Intraspecific crosses were confirmed by the presence of heteromeric allozymes. To determine single and joint segregation of biochemical loci, 84 single-spore-derived offspring from a confirmed hybrid sporocarp with five heterozygous loci were analyzed. Mendelian segregation was verified for the five loci. One linkage (Np withGpt,r=0.0) was found among 10 pairwise comparisons for joint segregation. The basidiospores ofV. volvacea are haploid and homokaryotic isolates may mate to yield heterokaryons.     

Response to imazapyr and dominance relationships of two imidazolinone-tolerant alleles at the <Emphasis Type="Italic">Ahasl1</Emphasis> locus of sunflower

Imisun and CLPlus are two imidazolinone (IMI) tolerance traits in sunflower (Helianthus annuus L.) determined by the expression of different alleles at the same locus, Ahasl1-1 and Ahasl1-3, respectively. This paper reports the level of tolerance expressed by plants containing both alleles in a homozygous, heterozygous and in a heterozygous stacked state to increasing doses of IMI at the enzyme and whole plant levels. Six genotypes of the Ahasl1 gene were compared with each other in three different genetic backgrounds. These materials were treated at the V2–V4 stage with increasing doses of imazapyr (from 0 to 480 g a.i. ha^–1) followed by an assessment of the aboveground biomass and herbicide phytotoxicity. The estimated dose of imazapyr required to reduce biomass accumulation by 50% (GR₅₀) differed statistically for the six genotypes of the Ahasl1 gene. Homozygous CLPlus (Ahasl1-3/Ahasl1-3) genotypes and materials containing a combination of both tolerant alleles (Imisun/CLPlus heterozygous stack, Ahasl1-1/Ahasl1-3) showed the highest values of GR₅₀, 300 times higher than the susceptible genotypes and more than 2.5 times higher than homozygous Imisun materials (Ahasl1-1/Ahasl1-1). In vitro AHAS enzyme activity assays using increasing doses of herbicide (from 0 to 100 μM) showed similar trends, where homozygous CLPlus materials and those containing heterozygous stacks of Imisun/CLPlus were statistically similar and showed the least level of inhibition of enzyme activity to increasing doses of herbicide. The degree of dominance for the accumulation of biomass after herbicide application calculated for the Ahasl1-1 allele indicated that it is co-dominant to recessive depending on the imazapyr dose used. By the contrary, the Ahasl1-3 allele showed dominance to semi dominance according to the applied dose. This last allele is dominant over Ahasl1-1 over the entire range of herbicide rates tested. At the level of enzymatic activity, however, both alleles showed recessivity to semi-recessivity with respect to the wild-type allele, even though the Ahasl1-3 allele is dominant over Ahasl1-1 at all the herbicides rates used.     

High Polymorphism and Autotetraploid Origin of the Rare Endemic SpeciesOxytropis chankaensisJurtz. (Fabaceae) Inferred from Allozyme Data

Using starch electrophoresis, we examined 19 enzyme systems presumably controlled by 35 loci in the rare endemic tetraploid speciesOxytropis chankaensisJurtz. (Fabaceae). Electrophoretic patterns and their genetic interpretation are presented. The isozyme data suggest tetrasomic inheritance in O. chankaensis. Three or four alleles at a particular locus were found in a number of individual plants, which indicate the autotetraploid origin of this species. Seventeen loci were shown to be polymorphic. As reliable gene markers for population systems, we recommend highly active polymorphic systems showing good allozyme separation (Ce-2, Gpi-2, Gpt-2, Idh-2, Lap, Mdh-2, and Mdh-3). Parameters of allozyme variability proved to be very high for a rare species with a restricted range: P = 48.6%, A = 2, A _p = 3.06, H _ob = 0.173.