Drosophila lactate dehydrogenase. Functional and evolutionary aspects.

The functional characteristics of homogeneously purified LDH were studied in the eight D. melanogaster species subgroup at two different growing temperatures (14 degrees C, 25 degrees C). The Vmax, kcat, Vmax/KeNAD.KmLac and Kcat/KsNAD KmLac values detected at the permissive growing temperature of 25 degrees C, were found to converge with the consensus phylogeny of these species which consists of two (melanogaster, yakuba) complexes. This scheme, also verified by the Micro-Complement Fixation (MC'F) method in another study in our Laboratory, substantiates the connection between the enzyme function and the phylogeny of these species. We propose that the major variation of the Ldh gene in this subgroup has arisen prior to the first species divergence, the final result of which is the eight sibling species. On the other hand, the variable catalytic differentiation observed at the restrictive temperature of 14 degrees C may enrich the species with hidden adaptive possibilities.

Distribution of P and hobo mobile elements in environmentally manipulated long-term Drosophila melanogaster cage populations.

The copy number and the chromosome positions of the P and hobo insertions were determined by means of in situ hybridization to polytene chromosomes, in five long-term Drosophila melanogaster cage populations kept for 18 years under different culture conditions (temperature and relative humidity). The analysis revealed that the copy number of both P and hobo elements were similar between the populations kept under the same culture conditions and significantly different among the populations maintained under different culture conditions. A tendency for similar distribution of these elements along the major chromosome arms was also observed in the populations of the same environmental manipulation. The distribution of the insertions along the chromosomes was not random for both the P and hobo elements; sites with high insertion frequencies were found (hot spots of occupation). Some of them were common in all cage populations while others were characteristic of the populations kept under the same conditions. Finally, fixed sites of occupation were also observed in all populations and refer mostly to hobo distribution. The data are discussed on the basis of the possible involvement of the P and hobo elements, in some way, to the adaptation process and speciation.

A three-season comparative analysis of the chromosomal distribution of P and hobo mobile elements in a natural population of Drosophila melanogaster.

An analysis on the chromosomal distribution of P and hobo elements in a Greek natural population extending over three seasons showed that the P elements were more abundant in the population than hobos. The copy number distribution per chromosome arm was in general random. The X chromosome had more P copies and the 3R arm more hobos in all three collections. Significant seasonal differences were not observed for these two elements in relation to the total number of insertions per haploid genome. There were, however, certain seasonal differences. They involved the copy number variability, the intra-arm distribution, the distribution along the chromosomes, and the spread and occupancy frequencies. There were no significant differences between the copy numbers of the two elements carried by the standard and the corresponding inverted regions for a number of inversions found in the population. Finally, three out of the five cosmopolitan inversions were found to have hobo insertions at or very near the one of the two breakpoints. Three out of the total had P insertions at or very near the one of the two breakpoints in some squashes and two of the three endemic inversions had a hobo insertion at or very near the one breakpoint, while the third had a P insertion.

Seasonal analysis of 23.5 MRF (hobo) and P-M hybrid dysgenesis determinants in a Greek natural population of Drosophila melanogaster.

Genetic analysis of 23.5 MRF (hobo) and P-M hybrid dysgenesis determinants in a Greek natural population in six collections over 24 months, showed the existence of hobo activity in the population at rates higher than P activity. Moreover, seasonal differentiation in hobo GD-sterility potential and hobo repressor abilities were observed. The P activity was low in the population but some tendency for seasonal differentiation of the cytotype was detected. The two systems operate independently in nature. Analysis of isofemale lines, established from inseminated wild-caught females, showed rapid differentiation of their hybrid dysgenesis determinants in the laboratory. This shows that results obtained from isofemale lines do not necessarily reflect the original population structure. The seasonal differentiation may be correlated with seasonal environmental factors, and may be attributed to differences in structure and function of the elements that consequently affect their regulation and transposition.

The effect of metals and alcohol on sexual isolation in Drosophila melanogaster.

Strong sexual isolation established between D. melanogaster long-term cage populations (originated from common parents and being under selection pressure since 1972) is maintained (with a tendency to increase) for twelve years after the origin of the populations. The sexual isolation is also maintained when the populations are kept in common conditions for about two years, while it dramatically decreases when the populations live on a food medium supplemented with strong chemical selective factors, such as various metals or ethanol. Seasonal or geographical studies of sexual isolation between natural and our cage populations did not reveal significant deviation from random matings. The genetic nature of sexual isolation is discussed.

Adaptation of Drosophila enzymes to temperature--V. Heat shock effect on the malate dehydrogenase of Drosophila melanogaster.

Drosophila melanogaster cMdh allozymic variants (MdhF MdhF/S, MdhS) were subjected to heat shock (33 degrees C/30 min----40 degrees C/30 min). This stress increases differentially MDH specific activity, with the cMdhF strain showing greater response as compared with the cMdhS one; heterozygotes exhibited generally intermediate values. Correlative differences were also revealed for some catalytic properties (Vmax, Vmax/Km ratio, thermostability) of the cMDH; this is not true for the mMDH. The catalytic behavior of the enzyme is correlated with the differential survival of the cMdh variants, with the cMdhF showing again higher survival than the cMdhS one, a fact which seems to contribute to temperature adaptation of D. melanogaster.

Indirect thermal selection in Drosophila melanogaster and adaptive consequences.

Short-term indirect selection in Drosophila melanogaster for heat-sensitivity and heat resistance resulted in two strains, one heat sensitive and another heat resistant, and correlated responses were found for the rate of heat shock protein synthesis, behavioral patterns (asymmetrical sexual isolation) and fitness components (fecundity, fertility, viability, developmental time), as well as for several enzyme activities (MDH, G-6-PDH, ADH, ACHE). These responses associated with temperature selection may reflect the effects of differential inbreeding depression caused by homozygosity of temperature sensitive mutations with different pleiotropic effects. Selection even of a very short duration can induce significant adaptive and evolutionary changes.

Drosophila lactate dehydrogenase: developmental aspects.

Partially purified lactate dehydrogenase (LDH) from third-instar larvae displays two bands (one major and one minor) on polyacrylamide gels. Analogous preparations from pupae and adults exhibit three LDH-staining bands (one major and two minor) in a similar pattern. The migration of the major band is similar for larvae, pupae, and adults, while the two minor LDH bands of pupae and adults migrate more slowly than the minor larval band. It has been shown that larval LDH incubated with beta-nicotinamide adenine dinucleotide exhibits two additional minor bands with an electrophoretic mobility similar to that of the minor bands of both pupae and adults. The intensity of the minor larval LDH band (exhibited also by untreated preparations) is drastically reduced. This fact indicates that the life-cycle stage-dependent LDH isozymic distribution is possibly due to a posttranslational effect(s). Highly purified LDH from larvae, pupae, or adults, obtained by an affinity chromatography procedure, displays just one dispersed band, located in the area between the band 5 and the band 6 exhibited by crude extract preparations. These data, in combination with the lack of difference in catalytic properties among enzymes from larvae, pupae, and adults, suggest that LDH synthesis is controlled by the same single structural gene at all developmental stages.

Non-Mendelian Inheritance of "Heat-Sensitivity" in DROSOPHILA MELANOGASTER.

Non-Mendelian inheritance was revealed for the "heat-sensitivity" character of the poikilothermic insect Drosophila melanogaster. Genetic analyses were performed on heat-sensitive (S, S(1)) strains, derived through indirect selection, and on stocks constructed through extensive chromosomal and cytoplasmic substitutions between strains obtained from two replicate cage populations. The populations were kept for about 7 years under different temperatures (14 degrees -25 degrees ) and exhibited different survival. We conclude that the character studied is quantitative, responds to selection pressure and is transmitted through the maternal cytoplasm, while nuclear genes modify its expression.

Drosophila lactate dehydrogenase: molecular and genetic aspects.

(LDH) obtained from larvae of Drosophila melanogaster was purified to homogeneity by affinity chromatography on oxamate-Sepharose. The purification procedure is simple to operate and gives a homogeneous preparation in a good yield (34.86%) after only two steps. Utilizing the homogeneous LDH preparation, an attempt was made to characterize the LDH molecule. Thus, it was found that the N-terminal amino acid is isoleucine, and the enzyme is tetrameric and composed of four identical subunits of apparent molecular weight 38,000, suggesting that it is controlled by a single gene. Homogeneous LDH preparations exhibit one band on neutral acrylamide gels when the substrate is either DL-lactic acid or L-(+)-lactate. The optimum temperature is 45 degrees C for the purified enzyme and 40 degrees C for the crude homogenate. The Km values for pyruvate and NADH are 0.154 and 0.027 mM, respectively, while the Km values for lactate and NAD are 29.4 and 1.33 mM, respectively. A discontinuity in the Ea slope was observed at a transition temperature of 30 degrees C. The Ea value between 20 and 30 degrees C was calculated as 12.06 kcal/mol, while between 30 and 45 degrees C the Ea value was 4.01 kcal/mol. This evidence, together with other observations reported in the literature, suggests that the LDHs of invertebrates and vertebrates have arisen by divergent evolution from a common ancestral gene.

Mating propensities and variations in enzyme activities in long-term cage populations of Drosophila melanogaster.

Environment-dependent reproductive isolation was established between cage populations (Bs) of Drosophila melanogaster originated from a Greek natural population (summer 1973) and maintained for about five years under different diets (poor-rich). The detected deviation from random mating involved no homogametic or heterogametic preference but rather, a significantly increased activity of males from populations maintained on the rich food medium. This observation indicates that the male parental investment is not negligible and under certain conditions sexual isolation can be a function not only of female behavior but also of male behavior. Differences also were found in various enzyme activities on the inter- and intra-population levels. Given those observations as well as the observed different behavioral patterns of Bs and Cs-Ds populations 19, a preliminary attempt was made to associate adaptive evolution with differences in enzyme activities. The differences in enzyme activities between populations reared on different media are not due to allozymic differences. It also was shown that in some populations environmental effects do not always elicit differences in enzyme activity. It was concluded, therefore, that the observed variations were the result of environmental effects interacting with modifier genes.

Enzyme specificity: "pseudopolymorphism" of lactate dehydrogenase in Drosophila melanogaster.

An electrophoretic variant in the LDH (L-lactate:NAD oxidoreductase, E.C.1.1.1.27) of Drosophila melanogaster was observed on starch (or polyacrylamide) gels. This variant was found to exhibit an identical isozymic pattern (three isozymes with a decreasing staining density) on starch gel and map position as the Adh locus. On the other hand, anodal polyacrylamide gel electrophoresis in crude extracts has shown LDH to consist of nine bands and ADH of four bands. We have shown than ADH (Alcohol: NAD oxidoreductase, E.C.1.1.1.1) also oxidizes L(+)-lactate or D(-)-lactate with the NAD, while LDH oxidizes ethanol. By using various genetic and biochemical techniques, we have shown that the observed Ldh electrophoretic variant was not a real one and could be attributed to the presence of ADH. We have called this phenomenon "pseudopolymorphism," and the problem of enzyme specificity has been examined. The appearance of a band in an assay using lactic acid as a substrate is not sufficient evidence for the presence of LDH. Hence, caution is called for before characterizing an electrophoretic band on a gel as being equivalent to the presence of a genetic locus. Out of the nine electrophoretic zones of activity observed on polyacrylamide gel (or out of the six previously observed) using crude extract, only two (one major and one minor) belong to LDH, as revealed by purified enzyme preparations. Furthermore, purified LDH exhibits activity in two bands on starch gel (out of three observed in crude extracts), which appear in different positions as compared with those of ADH. Finally, one band which responds to the presence of D(-)-lactate but not to L(+)-lactate has been revealed.

Behavioral and biochemical defects in temperature-sensitive acetylcholinesterase mutants of Drosophila melanogaster.

Temperature-sensitive (ts) mutants of the Ace gene, which codes for acetylcholinesterase (AChE) in Drosophila melanogaster, were analyzed for defects in viability, behavior and function of the enzyme. The use of heat-sensitive and cold-sensitive mutations permitted the function of AChE in the nervous system to be analyzed temporally. All ts mutations were lethal, or nearly so, when animals expressing them were subjected to restrictive temperatures during late embryonic and very early larval stages. Heat treatments to Ace-ts mid- and late larvae had little effect on the behavior of these animals or on the viability or behavior of the eventual adults. Heat-sensitive mutants exposed to nonpermissive temperatures as pupae, by contrast, had severe defects in phototaxis and locomotor activity as adults. AChE extracted from adult ts mutants that had developed at a permissive temperature were abnormally heat labile, and they had reduced substrate affinity when assayed at restrictive temperatures. However, enzyme activity did not decline during exposure of heat-sensitive adults to high temperatures even though such treatments caused decrements in phototaxis (29 degrees) and, eventually, cessation of movement (31 degrees). The cold-sensitive mutant also produced readily detectable levels of AChE when exposed to a restrictive temperature during the early developmental stage when this mutation causes almost complete lethality. We suggest that the relationship among the genetic, biochemical and neurobiological defects in these mutants may involve more than merely temperature-sensitive catalytic functions.

Adaptation of Drosophila enzymes to temperature. III. Evolutionary conservation in mitochondrial enzymes.

The evolutionary behavior of two mitochondrial enzymnes (L-glycerol 3-phosphate:cytochrome c oxidoreductase E.C.1.1.1.95, alpha GPO, and L-malate: NAD+ oxidoreductase, E.C.1.1.1.37, m-MDH) obtained from several temperate and tropical Drosophila species was examined by comparing their catalytic properties, which related to temperature (Km-Ea-Q10-Thermostability). Mitochondrial alpha GPO or m-MDH obtained either from template or from tropical species was found to exhibit similar catalytic properties while for both cytosolic enzymes, the alpha GPDH and s-MDH, Km patterns were similar among species from the same thermal habitat and different thermal habitats. In combination with other observations reported in the literature these facts support the view that the function, and probably the structure, of mitochondrial enzymes are better conserved in evolution than those of the corresponding enzymes found in the cytosol. It is proposed that the relative invariance of the mitochondrial enzymes structure is probably linked to a necessary relative invariance of molecular interactions inside the mitochondrion.