Quantitative subunit hybridization of drosophilaα-Glycerophosphate dehydrogenase

Summary The dimeric enzyme,-Glycerophosphate dehydrogenase, was purified from eight Drosophila species by the method of Collier et al. (1976). The enzymes were inactivated at high pH and the conditions sufficient for reactivation were established. Electrophoretic patterns of reactivated-glycerophosphate dehydrogenases which were mixed following inactivation of two species' enzymes, demonstrate that high pH dissociates the enzyme into its constituent subunits and reactivation involves subunit reassociation. Twenty interspecific combinations of dissociated enzymes were allowed to reassociate, and the amounts of both heterospecific and homospecific enzyme activity and protein were determined by densitometry. In all 20 tests there were no differences between observed and expected heterospecific:homospecific enzyme ratios. These results are consistent with the very slow rate of evolution of this enzyme in the family Drosophilidae (Collier and MacIntyre, 1977).     

Theoretical considerations of the sensitivity of quantitative subunit hybridization

Summary The theoretical basis of the quantitative subunit hybridization technique and its ability to measure evolutionary amino acid substitutions is examined. Homospecific:heterospecific enzyme ratios found after subunit reassociation depend upon K_1.2, the equilibrium constant for the dissociation of the heterospecific enzyme. It is shown that if this constant is near the geometric mean of the two homospecific enzyme dissociation constants, as it should be in enzymes whose subunits pair isologously, the quantitative subunit hybridization method will not detect most changes in the subunit contact regions of homologous proteins.     

Restriction enzymes do not play a significant role in Haemophilus homospecific or heterospecific transformation.

Competent Haemophilus influenzae Rd recipients, either as phage HP1 restricting (r+) or nonrestricting (r-) nonlysogens or defective lysogens, were exposed to deoxyribonucleic acids from various wild-type phage HP1 lysogenic H. influenzae serotype strains (non-encapsulated derivatives of serotypes a,b, c, d, and e), to DNA from lysogenic Haemophilus parahaemolyticus, and to DNA from modified and nonmodified phage HP1. Transformation of antibiotic resistance markers and of prophage markers in homospecific crosses was observed to be unaffected by the recipient restriction phenotype, whereas the transfection response was much reduced in r+ recipients. Heterospecific transformation of prophage markers was reduced by only 80 to 90%, whereas antibiotic resistance marker transformation was 1,000 to 10,000 times lower. Heterspecific transfection was at least 100 times lower than homospecific transfection in both r+ and r- recipients. The general conclusion is that neither class I nor class II restriction enzymes affect significantly the transformation efficiency in homospecific and heterospecific crosses. The efficiency of heterospecific transformation may depend mainly on the deoxyribonucleic acid homology in the genetic marker region.     

Influence of the Queen on Worker Behaviour and Queen Recognition Behaviour in Ants

We investigated the influence of the queen on worker behaviour in ants and their queen recognition behaviour. Queenless and queenright homo- and hetero-specific groups were created using three Myr-micinae species (Manica rubida, Myrmica rubra and Myrmica ruginodis). In homospecific groups, the presence of a queen contributed to an increase in the brood care, which had an effect on all other tasks of the group. In heterospecific groups, the presence of a queen led workers to care more for the conspecific brood. The queen appears to be a factor in the maintenance of both behavioural characteristics of the workers and the organization of the colony. The absence of the queen revealed some behavioural differences between species compared and populations that did not occur in queenright groups. Attractiveness tests were also conducted on these colonies. In homospecific queenright groups, workers were attracted both by unfamiliar conspecific and allospecific queens, but they were attracted more by the former. Therefore, queens appear to emit volatile pheromones which have a non-species-specific and a species-specific attractant effect. In heterospecific groups, workers were attracted more by an unfamiliar queen of the familiar species (even if allospecific) than by an unfamiliar conspecific queen, suggesting the importance of early social experience for the discrimination behaviour of adults.     

Immunological comparison of enzymes of the β-ketoadipate pathway

-Carboxy-cis,cis-muconate lactonizing enzyme and -carboxymuconolactone decarboxylase catalyze sequential reactions in the -ketoadipate pathway, the subunit sizes of the enzymes from Pseudomonas putida, biotype A, are 40000 and 13000, respectively. The cross reaction of antisera prepared against the enzymes was tested with the isofunctional enzymes formed by representatives of other bacterial species. Despite the differences in the subunit sizes of the enzymes, the antisera revealed the same general pattern: cross reaction was observed with the corresponding enzymes formed by other strains in the fluorescent Pseudomonas RNA homology group I and generally was not observed with enzymes from other Pseudomonas species or from other bacterial genera. Exceptions were provided by representatives of Pseudomonas cepacia. Members of this species are classified outside the fluorescent Pseudomonas RNA homology group. Nevertheless, the -carboxymuconolactone decarboxylases from these organisms formed precipitin bands with antisera prepared against the corresponding enzyme from P. putida, biotype A; the lactonizing enzymes from the two species did not appear to cross react. Immunodiffusion experiments with -carboxymuconolactone decarboxylase indicated that a common set of antigenic determinants for the enzyme is conserved among strains that have been classified together by other criteria; the relative immunological distances of the decarboxylases of each taxon from the reference P. putida, biotype A, enzyme were indicated by spurring patterns on Ouchterlony plates. These results suggested that the interspecific transfer of the structural gene for the enzyme is not a common event in Pseudomonas.Non-Standard Abbreviations CMLE -carboxy-cis,cis-muconate lactonizing enzyme (EC 5.5.1.2) - CMD -carboxymuconolactone decarboxylase (EC 4.1.1.44) - MLE cis,cis-muconate lactonizing enzyme (EC 5.5.1.1) - MI muconolactone isomerase (EC 5.3.3.4) Dedicated with affection and admiration to Professor R. Y. Stanier on his 60th birthday     

Genetic Integration in the Heterospecific Transformation of Haemophilus influenzae Cells by Haemophilus parainfluenzae Deoxyribonucleic Acid

The in vivo chemical linkage of Haemophilus parainfluenzae deoxyribonucleic acid (DNA) with the H. influenzae genome has been found to occur at a much higher level than is suggested by the low efficiency of the heterospecific transformation of an antibiotic resistance marker. This linkage, about 60% of the level with homospecific DNA, was found to involve alkali-stable bonding. The amount of host DNA label released (about 60%) was about the same as that released during homospecific transformation. Also, over 60% of the H. influenzae cells adsorbing H. parainfluenzae DNA could not form colonies upon plating. This lethality of the heterospecific transformation was not immediate but followed considerable metabolic activity of the host cells. These data are presented to show that the "limited-pairing" hypothesis may be only a partial explanation for the low efficiency of heterospecific transformation. Another hypothesis is presented which takes into account the lethal effect of this kind of transformation.     

Species recognition in wild-caught,laboratory-reared and cross-fostered Peromyscus californicus and Peromyscus eremicus (Rodentia,Cricetidae)

Experiments were performed to compare homospecific and heterospecific species choice in two closely related species of white-footed mice, Peromyscus californicus and P. eremicus. Both species significantly chose the homospecific stimulus animal. Significant homospecific choice was made by mice from sympatric but not from allopatric populations. Reciprocal cross-fostering between the two species resulted in significant choice for the heterospecific (foster) species by P. eremicus, and random choice by cross-fostered P. californicus. Laboratory-reared controls chose significantly for the homospecific chamber. No significant difference in choice performance was demonstrated between males and females, even when the oestrus stages of the females (both stimulus and test animals) were statistically controlled. A comparison of different test durations and temporal regimes of data collection was performed and 90 min was found to be the most efficient experiment duration with our apparatus.     

Relation of Macromolecular Synthesis in Streptococci to Efficiency of Transformation by Markers of Homospecific and Heterospecific Origin

In previous studies with Streptococcus sanguis and S. pneumoniae as recipients and donors of transforming deoxyribonucleic acid (DNA), it was found that heating recipients just prior to exposure to DNA caused an increase in the number of transformants induced by heterospecific DNA relative to that induced by homospecific DNA. In the present studies, S. sanguis recipients were found to recover from this effect of heat (48 C, 15 min) when incubated at 37 C before exposure to DNA. Inhibitors of nucleic acid synthesis, such as rifampin, 5-fluorodeoxyuridine, actinomycin, and p-hydroxyphenylazo-uracil, but not inhibitors of protein synthesis, such as chloramphenicol and erythromycin, prevented recovery from the effect of heat. Inhibitors of nucleic acid synthesis caused changes in unheated cells similar to those observed with heat treatment; these changes included increased transformability by genetically hybrid DNA and by low-efficiency markers in homospecific DNA. The effect of a combination of heat and inhibitors on transformation by heterospecific DNA was greater than when single treatments were used. The most effective inhibitor used alone was rifampin: in treated recipient cells, the yield of transformants produced by a given amount of irreversibly bound heterospecific DNA was increased without a significant change in the yield of transformants produced by bound homospecific DNA. A cell being doubly transformed by homospecific and heterospecific DNA was enhanced specifically in its transformability with the latter as a consequence of rifampin treatment. Treatment with rifampin also increased co-transformation by linked heterospecific markers. The period during which recipient cells were sensitive to the effects induced by rifampin and fluorodeoxyuridine lasted from 10 to 20 min after DNA uptake.     

Vibratory Communication and its Relevance to Reproductive Isolation in two Sympatric Stink Bug Species (Hemiptera: Pentatomidae: Pentatominae)

Communication is in phytophagous stink bugs of the subfamily Pentatominae related to mating behavior that among others includes location and recognition of the partner during calling and courting. Differences in temporal and frequency parameters of vibratory signals contributes to species reproductive isolation. Chinavia impicticornis and C. ubica are two green Neotropical stink bugs that live and mate on the same host plants. We tested the hypothesis that differences in temporal and spectral characteristics of both species vibratory signals enable their recognition to that extent that it interrupts further interspecific communication and copulation. To confirm or reject this hypothesis we monitored both species mating behaviour and recorded their vibratory songs on the non-resonant loudspeaker membranes and on the plant. The level of interspecific vibratory communication was tested also by playback experiments. Reproductive behavior and vibratory communication show similar patterns in both Chinavia species. Differences observed in temporal and spectral characteristics of female and male signals enable species discrimination by PCA analyses. Insects that respond to heterospecific vibratory signals do not step forward to behaviors leading to copulation. Results suggest that species isolation takes place in both investigated Chinavia species at an early stage of mating behavior reducing reproductive interference and the probability of heterospecific mating.     

Role of DL α-lipoic acid in gentamicin induced nephrotoxicity

The effect of DL -lipoic acid on the nephrotoxic potential of gentamicin was examined. Intraperitoneal injection of gentamicin (100 mg/kg/day) to rats resulted in decreased activity of the glycolytic enzymes-hexokinase, phosphoglucoisomerase, aldolase and lactate dehydrogenase. The two gluconeogenic enzymes—glucose-6-phosphatase and fructose-1, 6-diphosphatase, the transmembrane enzymes namely the Na⁺, K⁺-ATPase, Ca²⁺-ATPase, Mg²⁺-ATPase and the brushborder enzyme alkaline phosphatase, also showed decreased activities. This decrease in the activities of ATPases and alkaline phosphatase suggests basolateral and brush border membrane damage. Decreased activity of the TCA cycle enzymes isocitrate dehydrogenase (ICDH), succinate dehydrogenase (SDH) and malate dehydrogenase (MDH), suggests a loss in mitochondrial integrity. These biochemical disturbances were effectively counteracted by lipoic acid administration. Lipoic acid administration by gastric intubation at two different concentrations (10 mg and 25 mg/kg/day) brought about an increase in the activity of the glycolytic enzymes, ATPases and the TCA cycle enzymes. The gluconeogenic enzymes however showed a further decrease in their activities at both the concentrations of lipoic acid administered. These observations shed light on the nephroprotective action of lipoic acid against experimental aminoglycoside toxicity and the protection afforded at 25 mg/kg/day of lipoic acid was noted to be higher than that at 10 mg level.     

Lysosomal enzymes in experimental allergic encephalomyelitis: Time course and evidence of the source

The lysosomal enzymes acid proteinase and -glucuronidase, were assayed in spinal cords of rats during the course of experimental allergic encephalomyelitis (EAE). Histological and histochemical examination was carried out versus controls, in selected areas of the same cords biochemically assayed, to look at the distribution of the lysosomal enzyme acid phosphatase. The biochemical assay showed a significant increase of the enzyme activities during the disease and the increase was significantly correlated with the intensity of the disease. The distribution in the nervous tissue of the increase in acid phosphatase activity observed in animals with EAE, suggests that endogenous nervous cells may contribute to the lysosomal enzyme increase in EAE.     

Post-pollination prefertilization drops affect germination rates of heterospecific pollen in larch and Douglas-fir

Pollen of larch (Larix?×?marschlinsii) and Douglas-fir (Pseudotsuga menziesii) was used in homospecific and heterospecific crosses. Germination of heterospecific pollen in ovulo was reduced in post-pollination prefertilization drops. This provides evidence of selection against foreign pollen by open-pollinated exposed ovules in these two sister taxa, which share the same type of pollination mechanism. Of the other prezygotic stages in pollen-ovule interactions, uptake of pollen by stigmatic hairs did not show any selection. Pollen tube penetration of the nucellus was similar for hetero- and homospecific pollen tubes, but heterospecific tubes only delivered gametes in one cross. To test for differences in the post-pollination prefertilization drops of each species, drops were gathered and analysed. Glucose and fructose were present in similar amounts in Douglas-fir and larch, while sucrose was found in larch only. Other carbohydrates such as xylose and melezitose were species-specific. In P. menziesii, sucrose is absent due to its conversion to glucose and fructose by apoplastic invertases. In contrast, Larix?×?marschlinsii drops have sucrose because they lack apoplastic invertases. The presence of invertase activity shows that the composition of gymnosperm post-pollination prefertilization drops is not static but dynamic. Drops of these two species also differed in their calcium concentrations.     

Changes of species specific cuticular hydrocarbon profiles in the cockroaches Nauphoeta cinerea and Leucophaea maderae reared in heterospecific groups

The cuticular hydrocarbon profiles of the cockroaches Nauphoeta cinerea and Leucophaea maderae are species-specific when maintained in homospecific rearings. When individuals were reared in mixed species colonies, they initially remained in homospecific groups under different shelters. However, after 14 days they formed one heterospecific group with cuticular profiles showing characteristics of both species. When individuals were returned in monospecific rearings, their cuticular hydrocarbon profiles returned to species-specific ones within 3 weeks.     

Courtship behavior and sexual isolation between Drosophila auraria and D. triauraria in darkness and light

Sexual isolation between two species of the Drosophila auraria complex, D. auraria and D. triauraria is different in darkness and light. In darkness there is complete isolation while in light it is only partial. The sensory bases of these differences were investigated by behavioral studies in darkness and light. In darkness there was no normal courtship sequence but males of both species displayed attempted copulation to homospecific and heterospecific females, as well as homospecific males. After a few hours together, homospecific copulations were observed after females displayed a characteristic posture: spreading the wings. Such an acceptance posture, also observed in other Drosophila species, was probably released by the wing vibration of a homospecific male. In light, visual signals alone are able to elicit male's orientation and following, whether flies are housed together or physically separated. Intense homosexual courtships were observed. All these data suggest that males have a low discrimination ability and females play the main role in sexual isolation.     

Liver phosphorylase phosphatase

Summary Recent progress in studies on the properties and regulation of liver phosphorylase phosphatase can be divided into four stages. First, isolation from multiple molecular forms of phosphorylase phosphatase, of a single form of catalytic subunit (Mr = 32 000-35 000) which is active toward phosphorylase a and also toward a variety of protein substrates phosphorylated by either cyclic AMP-dependent protein kinase or other protein kinases. This was achieved by rather drastic procedures such as treatment with 80% ethanol at room temperature, incubation with 6 M urea, freeze-thawing in the presence of 0.2 M mercaptoethanol, or digestion by trypsin. These treatments caused concomitantly large enhancement of phosphorylase phosphatase activity, and the hypothesis was proposed that an inactive form of phosphorylase phosphatase existed as complexes of a catalytic subunit and inhibitory proteins. Second, it was discovered that liver and muscle extracts contain trypsin-labile proteins which, after heating at 90 °C, inhibited the catalytic subunit of phosphorylase phosphatase. Two types of protein inhibitors were identified: inhibitor-I was phosphorylated and activated by cyclic AMP-dependent protein kinase, whereas inhibitor-2 was not phosphorylated. Although inhibitor-1 has been implicated in hormonal regulation of glycogen metabolism in skeletal muscle, a similar role of protein inhibitors in the regulation of phosphorylase phosphatase in the liver has not been demonstrated and the physiological role of the inhibitor is questionable.Third, it has been demonstrated that liver phosphorylase phosphatase is reversibly inactivated and regulated by glutathione disulfide (GSSG) at the catalytic subunit level. Liver phosphorylase phosphatase contains, per mole of catalytic subunit, two sulfhydryl groups, one of which reacts with GSSG to form mixed disulfide with consequent inactivation of the enzyme. The inactivated enzyme can be reactivated by glutathione(GSH) or other sulfhydryl compounds through a reverse reaction. Injection of GSSG into the portal vein of rabbits caused a rapid increase in phosphorylase-a activity in the liver, suggesting that GSSG is involved in regulation of phosphorylase activity in vivo.Finally, current evidence suggests that liver phosphorylase phosphatase exists in the native state in a high molecular weight form which consists of the catalytic subunit and other regulatory subunits. One such enzyme species could be a 260 000-dalton protein composed of three different types of subunit, termed , and , or a 160 000-dalton protein composed of and subunits. The a subunit (Mr = 35 000) appears to be identical to the multifunctional catalytic subunit, whereas the (Mr = 69 000) and (Mr = 58 000) subunits are catalytically inactive but can modify the catalytic a subunit. It seems likely that the substrate specificity and catalytic activity of the subunit is considerably altered when it is part of larger complexes with other regulatory subunits ( and ). It has also been suggested that in addition to the native form of phosphorylase phosphatase, liver contains a considerably large amount of latent phosphorylase phosphatase, the catalytic activity of which could be revealed only by treatment with trypsin or ethanol.     

Enzyme activities in plasma, kidney, liver, and muscle of five avian species

Activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), creatine phosphokinase (CPK), and lactate dehydrogenase (LDH) were determined in plasma, kidney, liver, and muscle from five species of captive birds. Few differences occurred in plasma activities between sexes but considerable differences occurred between species. All five enzymes were detected in each of the tissues sampled. Relative enzyme activities in liver, kidney, and muscle were similar for each species. CPK activity was much higher in muscle than in liver or kidney and, of the five enzymes studied, may be the best indicator of muscle damage. Most of the other enzymes were more evenly distributed among the three tissues, and no organ-specific enzyme could be identified for liver or kidney. Because of interspecific variations in plasma enzyme activities, it is important to establish baseline values for each species to ensure accurate interpretation of results.     

Fate of heterospecific transforming DNA bound to Streptococcus sanguis.

The fate of 3H-labeled str-r fus-s DNA from Streptococcus pneumoniae, bound after a 1-min uptake to 14C-labeled str-s fus-r S. sanguis recipients, was followed by techniques previously developed for analyzing the fate of homospecific DNA. Heterospecific S. pneumoniae DNA was bound and formed complexes with recipient protein in a manner similar to that of homospecific DNA but transformed relatively poorly. The rate at which complexed heterospecific DNA becomes physically associated with recipient DNA, and at which donor markers are integrated into the chromosome, was slower than in the case of homospecific DNA. In addition, about half of the heterospecific donor counts initially bound in trichloracetic acid-insoluble form were gradually solubilized and released from the cell. The association of heterospecific DNA with the recipient chromosome was more unstable than that involving homospecific DNA, since only associations of the former type were largely dissociated by isolation and resedimentation. The donor DNA-containing material so dissociated had the same sedimentation properties as complexed heterospecific DNA before association, indicating that the complex of single-stranded donor DNA and recipient protein formed on uptake moves as a whole from its site of formation to synapse with the chromosome.     

Genetic Hybridization at the Unlinked THY and STR Loci of Streptococcus

The sanguis and pneumoniae species of Streptococcus were used as recipients in transformations from str+ to str-r and from thy- to thy+. The str-r mutations in the two species had been previously shown to be allelic. Homology of the thy- mutations in the two species was demonstrated in the similar phenotypic properties they conferred (death in the absence of thymidine, lack of thymidylate synthetase). The str and thy loci are unlinked in each species.--- When the two species are transformed by both homospecific and heterospecific DNA, the efficiency is always lower in the heterospecific cross. The efficiency of heterospecific transformation is considerably lower at the thy than at the str locus. DNA was extracted from recipients that had integrated markers of heterospecific origin. When such hybrid DNA is tested on the original recipient species, the heterospecific markers are usually as efficient as homospecific markers. When tested on the original donor species, however, the hybrid DNA is usually more efficient than heterospecific DNA. This is true for both thy and str transformation. -- -- Forty independent thy+ hybrids were obtained in the cross of sanguis thy- recipients with pneumoniae thy+ DNA. These hybrids fall into a number of classes based upon the relative efficiency with which their extracted DNA's are able to transfer the thy+ marker into pneumoniae thy- cells. The most efficient of these DNA's exhibits about 20% of the efficiency of homospecific pneumoniae thy+ DNA and three orders of magnitude greater efficiency than heterospecific sanguis thy+ DNA. Thus, very little of the inefficiency of heterospecific transformation of the thy locus is ascribable to a classic restriction mechanism. Rather, the wild-type thy+ loci in the two species appear to differ at multiple sites, and independent heterospecific transfers result in differential extents of integration of these sites. On this basis, the thy+ loci of the two species differ at a greater number of sites than do the respective str+ loci.     

Introgression of mtDNA in Urosaurus lizards: historical and ecological processes

Introgression of mtDNA appears common in animals, but the implications of acquiring a novel mitochondrial genome are not well known. This study investigates mito‐genome introgression between the lizard species Urosaurus graciosus, a thermal specialist, and U. ornatus, a species that occupies a wider range of thermal environments. As ectotherms, their metabolic rate is strongly influenced by the thermal environment; with mitochondria being linked to metabolic rates, overall energy budgets could be impacted by introgression. I use mitochondrial gene trees, inferred from Bayesian analyses of Cyt‐B and ND1 gene sequences, along with morphology and microsatellites from nineteen populations of these two species to address if the direction and location of mito‐nuclear discordance match predictions of introgression resulting from past population expansions. MtDNA is expected to move from resident species into expanding or invading species. Second, does having a heterospecific form of mitochondria impact body size, a trait strongly associated with fitness? Multiple independent introgression events of historic origin were detected. All introgression was unidirectional with U. ornatus‐type mtDNA found in U. graciosus parental type individuals. This result was consistent with population expansions detected in U. graciosus but not U. ornatus. Females with heterospecific mtDNA were significantly smaller than homospecific forms, and heterospecific males had a different relationship of body mass to body length than those with homospecific mtDNA. These changes indicate a potential selective disadvantage for individuals with heterospecific mitochondria and are consistent with the theoretical expectation that deleterious alleles are more likely to persist in expanding populations.