Variation in Biochemical Properties of Allozymes of Xanthine Dehydrogenase in DROSOPHILA PSEUDOOBSCURA

Twenty-six D. pseudoobscura strains isogenic for xanthine dehydrogenase alleles from Mesa Verde, Colorado, were tested for differences in the biochemical properties of different allelic forms of xanthine dehydrogenase. No significant differences in binding affinity (K_m) or substrate specificity of the enzyme were found. Significant variation among strains, in activity (V_max) and among electromorphs, as well as among strains, in thermolability was found. For the few strains tested, the activity and thermolability differences were shown to co-segregate with the electrophoretic mobility of the variant allele.     

Distinctions among Allelic Variants Associated with Chromosome 3 Inversions in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

Efforts were made to discriminate new genetic variants among electrophoretic alleles that are associated with chromosome 3 inversions of Drosophila pseudoobscura and D. persimilis. Apparent genetic similarities for electrophoretic alleles between these two species and among the common inversions they carry were reexamined by altering gel concentration and buffer pH. At the amylase locus, the 1.09 electrophoretic allele could be further separated into two allelic classes that differentiated the WT and KL arrangements. Similarly, the 0.84 electrophoretic allele was divided into two allelic classes, one characteristic of the Santa Cruz phylad arrangements, TL and SC, and the other found in strains of the Standard phylad arrangements and CH. Uncommon amylase alleles proved to be different alleles in the two species. No new allelic variants, however, could be found among strains with the amylase 1.00 allele, the commonest allele in the Standard phylad of both species. No major new allelic variation was detected for acid phosphatase-3 and larval protein-10 that revealed any further differentiation among species or inversions. Variation at all three loci in strains of the Bogota population remained genetically similar to variation in strains of mainland D. pseudoobscura.     

Genetic Heterogeneity within Electrophoretic "Alleles" of Xanthine Dehydrogenase in DROSOPHILA PSEUDOOBSCURA

An experimental plan for an exhaustive determination of genic variation at structural gene loci is presented. In the initial steps of this program, 146 isochromosomal lines from 12 geographic populations of D. pseudoobscura were examined for allelic variation of xanthine dehydrogenase by the serial use of 4 different electrophoretic conditions and a head stability test. The 5 criteria revealed a total of 37 allelic classes out of the 146 genomes examined where only 6 had been previously revealed by the usual method of gel electrophoresis. This immense increase in genic variation also showed previously unsuspected population differences between the main part of the species distribution and the isolated population of Bogotá population. The average heterozygosity at the Xdh locus is at least 72% in natural populations. This result, together with the very large number of alleles segregating and the pattern of allelic frequencies, has implications for theories of genetic polymorphism which are discussed.     

Hidden Electrophoretic Variation at the Xanthine Dehydrogenase Locus in a Natural Population of DROSOPHILA MELANOGASTER

Sixty-two isochromosomal lines of D. melanogaster were screened for cryptic electrophoretic variation at the xanthine dehydrogenase (XDH) locus. Sequential polyacrylamide vertical slab gel electrophoresis was performed using four electrophoretic criteria. A total of 15 classes of electromorphs were revealed. D. melanogaster appears to exhibit as much polymorphism at this locus as other extensively studied Drosophila species.--No evidence for loci on the X or second chromosomes which modified XDH mobility was found. Six of the electromorphs were mapped to the Xdh (ry) structural locus. Eight of the remaining nine classes exhibited mobility variation consistent with structural variation at the Xdh locus. The final class exhibited aberrant patterns and is under further study.     

Frequency-Dependent Selection at the PGM-1 Locus of DROSOPHILA PSEUDOOBSCURA

Frequency-dependent fitness was studied at the Pgm-1 locus of Drosophila pseudoobscura with respect to two fitness components: rate of development and larva-to-adult survival. The Pgm-1 locus is very polymorphic with only two alleles, Pgm-1¹⁰⁰ and Pgm-1¹⁰⁴, occurring at high frequencies. For each of these two alleles, 20 homozygous strains were obtained from a sample of 1,140 wild-inseminated females. First-instar larvae of the two genotypes were combined in a set of eight different frequencies: 0.0, 0.10, 0.25, 0.40, 0.60, 0.75, 0.90, and 1.0. Frequency-dependent fitness effects were observed for the two survival-related fitness components examined: larvae of the less common genotype develop faster and have a higher probability of survival than larvae of the more common genotype. The rate of survival at intermediate genotypic frequencies is similar to that in pure cultures. If selection acted solely as frequency-dependent effects on survival-related components of fitness, the equilibrium frequency of the Pgm-1¹⁰⁰ allele would be 0.615 for a two-genotype system, which fits an observed frequency range for this allele in nature between 0.55 and 0.71. Experimentally created linkage disequilibrium was excluded from the experiment by using a large number of independent strains. It is nevertheless possible that the frequency-dependent selection may not affect the Pgm-1 locus per se, but may reflect a linkage disequilibrium present in the natural population. Even if this were the case, the frequency-dependent selection could affect the frequency of the Pgm-1 alleles in nature.     

Genic Heterogeneity at Two Alcohol Dehydrogenase Loci in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

A sequential electrophoretic survey of the second chromosome loci, alcohol dehydrogenase-6 (Adh-6) and octanol dehydrogenase ( Odh), was performed on 147 isochromosomal lines of Drosophila pseudoobscura and 60 lines of its sibling species, D. persimilis. Gels run with a variety of acrylamide concentrations and buffer pH's revealed the presence of 18 alleles of Adh-6 in the two species, where only eight had been previously detected by conventional electrophoretic methods. Only two alleles were added with our techniques to the previous total of nine in both species at the largely monomorphic Odh locus. Both enzymes show a predominance of one allele, with the other variants being fairly rare. There was no evidence of increased genetic divergence between the two species, but we found a striking increase in differentiation of Adh-6 alleles between the main body of D. pseudoobscura populations and the conspecific isolate from Bogotá, Colombia. These results are compared with our previous surveys of xanthine dehydrogenase in these species and discussed in reference to theories of genic polymorphism.     

Density- and Frequency-Dependent Selection at the Mdh-2 Locus in DROSOPHILA PSEUDOOBSCURA

We have studied differences in the number of Drosophila pseudoobscura produced in a culture when the flies differ with respect to two alleles (F and S) at the Mdh-2 locus, which codes for a malate dehydrogenase enzyme. The studies were done at low and at high density in two- and three-genotype combinations (S/S, F/F and S/F), with one-genotype cultures as controls.——Density affects the fitness of the Mdh-2 genotypes. Different genotypes are differently affected, and the genotype of the competitors also makes a difference on the fitness of a given genotype. When three genotypes are present in a culture, particularly at high density, intergenotypic competition is less intense than intragenotypic competition at several frequency combinations. That is, there is "overcompensation": the three genotypes together exploit the environmental resources better than one genotype alone.—The fitness of the genotypes is frequency dependent in both two-genotype and three-genotype combinations. An inverse relationship between frequency and fitness is observed at high density. This may lead to a stable polymorphism, because the fitness of a genotype increases as its frequency decreases.—Forty independent strains, sampled from a natural population, were used in the experiments. This ensures that more than 95% of the variation present in the genome in the natural population is also present is the experimental cultures. It also ensures that the genetic background of the Mdh-2 alleles is randomized in the same way as it is in nature. However, the possibility remains that Mdh-2 alleles in nature are nonrandomly associated with alleles at closely linked loci. If linkage disequilibrium is present in the experiments because it exists in nature, then the observed effects (such as frequency-dependent selection) would affect the Mdh-2 locus in nature as well.     

Polymorphism for Dimerizing Ability at the Esterase-5 Locus in DROSOPHILA PSEUDOOBSCURA

Three new alleles are reported at the esterase-5 locus of Drosophila pseudoobscura. All three of these alleles are different from those previously reported in their ability to dimerize. One allele will not form heterodimers or homodimers and exists only as a monomer. A second allele does form heterodimers but will not form homodimers. The third allele forms both hetero- and homodimers as well as forming monomers. Estimates of the frequency of these alleles in a natural population are given. The existence of these is discussed with respect to recently proposed models for a molecular mechanism for heterosis.     

Genetic Limits of the Xanthine Dehydrogenase Structural Element within the Rosy Locus in DROSOPHILA MELANOGASTER

Experiments are described that provide an opportunity to estimate the genetic limits of the structural (amino acid coding) portion of the rosy locus (3:52.0) in Drosophila melanogaster, which controls the enzyme, xanthine dehydrogenase (XDH). This is accomplished by mapping experiments which localize sites responsible for electrophoretic variation in the enzyme on the known genetic map of null-XDH rosy mutants. Electrophoretic sites are distributed along a large portion of the null mutant map. A cis-trans test involving electrophoretic variants in the left- and right-hand portions of the map leads to the conclusion that the entire region between these variants is also structural. Hence most, if not all, of the null mutant map of the rosy locus contains structural information for the amino acid sequence of the XDH polypeptide. Consideration is given to the significance of the present results for the general problem of gene organization in higher eukaryotes.     

Gene Frequency Changes at the alpha-Amylase Locus in Experimental Populations of DROSOPHILA PSEUDOOBSCURA

The frequencies of alleles at the α-Amylase locus of D. pseudoobscura were followed in both large and small experimental populations. No evidence for balancing or directional selection was found, although our ability to detect weak selection is limited. The gene frequency changes in our experimental populations were consistent with the hypothesis of selective neutrality and genetic drift due to sampling error.     

Allelic Negative Complementation at the Abruptex Locus of DROSOPHILA MELANOGASTER

The mutations of the Abruptex locus in Drosophila melanogaster fall into three categories. There are recessive lethal alleles and viable alleles. The latter can be divided into suppressors and nonsuppressors of Notch mutations. The recessive lethals are lethal in heterozygous combination with Notch. As a rule the recessive lethals are lethal also in heterozygous combination with the viable alleles. Heterozygous combinations of certain viable alleles are also lethal. In such heterozygotes, one heteroallele is a suppressor of Notch and the other is a nonsuppressor. Other heterozygous combinations of viable alleles are viable and have an Abruptex phenotype. The insertion of the wild allele of the Abruptex locus as an extra dose (carried by a duplication) into the chromosomal complement of the fly fully restores the viability of the otherwise lethal heterozygotes if two viable alleles are involved. The extra wild allele also restores the viability of heterozygotes in which a lethal and a suppressor allele are present. If, however, a lethal and a nonsuppressor are involved, the wild allele only partly restores the viability, and the effect of the wild allele is weakest if two lethal alleles are involved. It seems likely that of the viable alleles the suppressors of Notch are hypermorphic and the nonsuppressors are hypomorphic. The lethal alleles share properties of both types, and are possibly antimorphic mutations. It is suggested that the locus is responsible for a single function which, however, consists of two components. The hypermorphic mutations are defects of the one component and the hypomorphic mutations of the other. In heterozygotes their cumulative action leads to decreased viability. The lethal alleles are supposed to be defects of the function as a whole. The function controlled by the locus might be a regulative function.     

Activity Variants of Acid Phosphatase-3 among Chromosome 3 Inversions of DROSOPHILA PSEUDOOBSCURA

Sexual selection is measured between two strains of Drosophila melanogaster: a wild strain and a strain mutant at the sepia locus. Frequency-dependent male mating was found to be successful, whereas the female genotype exerted no influence. The rarer the male genotype becomes, the greater is its mating success. A selection model is built for this behavior characteristic in which selection operates differently in the two sexes. The genetic consequencies of this model upon the maintenance of genetic polymorphism at the sepia locus are compared to experimental data from previous population cage studies. The fit obtained with this sexual selection model is compared to that of the larvel selection model previously investigated. A model composed of both sexual and larval components of fitness is presented. The role that each major selection component is expected to play in experimental populations as the gene frequency changes is discussed. Sexual selection leads to an equilibrium level higher than larval selection, and the combined model is very close to the experimental values.     

Developmental Changes in the Activity of Xanthine Dehydrogenase and closely Related Enzyme in Chick Tissues

Xanthine-oxidizing activities in the chick tissues were measured by a couple of assay procedures during development of chick embryo. With the usual assay using pterine and NAD⁺, no detectable level of XDH activity was observed in the liver and little in the duodenum before hatching, whereas an appreciable activity was detected in the kidney of chick embryo. When assayed with xanthine and dichlorophenol indophenol, an XDH-like enzyme activity was significantly detected in the embryonic liver, while no further enhancement of the activity was detected in the kidney and duodenum. Electrophoregrams obtained with samples from various developmental stages, followed by activity staining with tetrazolium dye, supported the above results and revealed that the embryonic XDH-like enzyme is not distinguishable from XDH of adult tissue in molecular size. This XDH-like enzyme, pre-existing in the liver before hatching, however, exhibited no cross reaction with antibody against intact XDH. The nature of this material was discussed in comparison with deflavinated XDH.     

Organization of the Rosy Locus in DROSOPHILA MELANOGASTER : Evidence for a Control Element Adjacent to the Xanthine Dehydrogenase Structural Element

From a collection of electrophoretic variants of XDH obtained from laboratory strains and natural populations, a stock was isolated that was associated with much greater than normal levels of XDH activity. Preliminary recombination experiments demonstrated that this character maps to the rosy locus. While a series of observations failed to relate this phenotype to alteration in the structure of the XDH polypeptide, kinetic and immunological experiments did succeed in associating this character with variation in number of molecules of XDH/fly. Large scale fine structure recombination experiments locate the genetic basis for this variation in number of molecules of XDH/fly to a site very close to, but definitely outside of, the genetic boundaries of the XDH structural information. Observations are described which eliminate the possibility that we are dealing with a tandem duplication of the XDH structural element. Turning to a regulatory role for this genetic element located adjacent to the XDH structural information, a simple experiment is described which demonstrates that it functions as a "cis-acting" regulator of the XDH structural element.