Thymidylate Synthetase in Mutants of DROSOPHILA MELANOGASTER

Thymidylate synthetase has been examined with respect to the normal pattern of activity throughout the development of the Oregon-R strain of Drosophila melanogaster. Large amounts of the enzyme are present in both the unfertilized and the fertilized eggs. A comparison of the ovarian thymidylate synthetase activity of the Oregon-R strain and the female sterility mutants, fs(2)B, fu, and fs(1)N, indicates variations in the activity of this enzyme in each strain. At four days of age, the ovarian-specific activity of the female sterility mutants is comparable to or less than that of the Oregon-R strain, but it is reduced at fifteen days of age. The enzyme activity per ovary is low in the fs(2)B strain but is similar in the Oregon-R, fu, and fs(1)N strains. When expressed as activity per organism, thymidylate synthetase declines after six to eight hours of development until the minimal level is reached in the late embryonic stage. Enzyme activity rises throughout the larval instars, reaching a maximum immediately after puparium formation. The activity decreases during pupation, but rises again during the first four days of adult life.     

Ecdysone titers and prothoracic gland activity during the larval-pupal development of Manduca sexta

The titer of ecdysone in whole animal extracts of Manduca sexta was determined by radioimmunoassay during the fifth (last) larval instar, pharate pupal development and pupation. A subtle peak in ecdysone concentration was noted at day 4 (just prior to the onset of the wandering stage) and a second and greater peak at day 8.5 (coincident with pharate pupal development). The titer fluctuations during development were a result of changes in tissue ecdysone and not of alterations in the ecdysone content of the gut. When prothoracic gland secretory activity was analyzed in vitro at the same stages, the most rapid rate of α-ecdysone secretion was shown to occur on day 7 (one day prior to the peak in whole-animal ecdysone concentration). An earlier peak in prothoracic gland activity may occur at day 4–5. Thin layer and gas-liquid chromatographic analyses revealed developmental changes in the ratio of β:α-ecdysone in hemolymph and whole-animal extracts. It is suggested that the steroid-hydroxylating capacity of the insect increases during the instar.     

RNA metabolism during metamorphosis of Drosophila hydei

Determinations of total protein and RNA at various times after the beginning of the pharate pupal stage of Drosophila hydei, revealed an increase in both substances during the first 25 hr and a sudden decrease thereafter until 52 hr. From this time on the total amount of both protein and RNA increases slightly until emergence of the flies at 160 hr after the beginning of the pharate pupal stage. A similar pattern of changes was recorded for the total radioactivity as well as the specific activity of RNA labelled with ³H-uridine after the injection of the isotope immediately before the beginning of the pharate pupal stage.The migration profile of RNA labelled with ³H-uridine during larval development, revealed that shortly after the onset of the pharate pupal stage an essentially normal larval pattern consisting of major fractions of 28, 18, 8 to 9, and 4 to 7 S RNA. At 52 hr only the low molecular weight RNA was present. The ‘normal’ pattern was restored at the time of emergence of the flies, indicating re-utilization of degradation products of previously labelled RNA.     

Thymidylate synthetase during synchronous nuclear division cycle and differentiation of Physarum polycephalum

Thymidylate synthetase and thymidine kinase activities in wild type strain M₃b and in thymidine kinase-deficient mutant TU63 of Physarum polycephalum are studied. Whenever nuclear division occurs in macroplasmodia of wild type, thymidine kinase and thymidylate synthetase activities sharply increase, although the increase of thymidylate synthetase activity is less pronounced than thymidine kinase activity. This is also true for other investigated nuclear divisions during the life cycle of P. polycephalum. It is shown for the first time that thymidylate synthetase is a periodically fluctuating enzyme during the naturally synchronous nuclear division cycle of P. polycephalum with a peak of specific activity in the S phase. In macroplasmodia, as well as after germination of microsclerotia of M₃b, thymidine kinase is the dominant enzyme, whereas at the time of the precleavage mitosis in sporulating macroplasmodia thymidylate synthetase is the predominant enzyme. This study describes and compares both dTMP-synthesizing enzymes during proliferation and differentiation of the same organism.     

Fatty acid synthetase content during development of the fly,Ceratitis capitata

Immunochemical titrations of different enzyme preparations from larval, pharate adult, and emerged adult Ceratitis capitata indicated that the changes in fatty acid synthetase activity during development of the insect are not related entirely to changes in the content of the enzyme. Changes in catalytic efficiency during larval and pharate adult development were clearly paralleled by the amounts of immunoprecipitate; however, the changes of enzyme activity with adult age were not correlated to the changes of enzyme content. Dietary manipulations of the larval stage of the insect Ceratitis capitata show the adaptive nature of the fatty acid synthetase. Fasting produced a clear decrease of activity and level of the enzyme from the larvae and refeeding restored practically normal values.     

Cyclic AMP phosphodiesterase activity during the development of the silkworm, Bombyx mori

Cyclic AMP phosphodiesterase activity has been demonstrated in homogenates of the silkworm, Bombyx mori, throughout larval, pupal, and pharate adult life. The enzyme exhibits a pH optimum at pH 8·0, and the enzyme activity was inhibited in the presence of caffeine and theophylline. The enzyme activity increases after hatching, and a relatively higher level of the activity is maintained through the larval stage. The activity, however, falls markedly before pupation and increases sharply once again before emergence.     

Gene-controlled incorporation of haemolymph protein into the ovaries of Bombyx mori

The haemolymph protein concentration in Bombyx mori decreases normally by about one-fourth during pharate adult development. In females homozygous for the small egg gene, the concentration of haemolymph protein remained constant throughout the pupal and pharate adult stages. The sm gene does not influence the synthesis of vitellogenic female protein of pupal and pharate adult haemolymph (FP). Normal ovaries transferred to the haemocoele of sm females undergo normal vitellogenesis. In the absence of normal alleles of sm, the ovaries encounter difficulties in the incorporation of FP into their oöcytes from pharate adult haemolymph. These results suggest that an active translocation mechanism is involved in the transfer of haemolymph protein into the ovaries.     

Enzymes of DNA biosynthesis in developing sea urchins. Changes in ribonucleotide reductase, thymidine, and thymidylate kinase activities.

The pattern of ribonucleotide reductase, thymidine kinase, and thymidylate kinase activities during development of Paracentrotus lividus eggs and the effect of actinomycin on these enzymatic activities have been studied. Ribonucleotide reductase activity is detectable, though at a low level, in the unfertilized egg; the activity increases sharply soon after fertilization and reaches a peak at the morula stage. Thereafter it decreases and remains at a lower level than that of the unfertilized egg. Actinomycin, at a concentration sufficient to inhibit messenger RNA (mRNA) synthesis does not affect the level of enzymatic activity, indicating that preexisting maternal mRNA is used for the synthesis of this enzyme. Thymidine kinase is present at a low level in the egg; it increases sharply after the hatching blastula until the pluteus stage. Actinomycin does not affect the enzyme activity from fertilization until blastula but prevents the increase in enzyme activity that is observed between blastula and pluteus. Thymidylate kinase activity shows an increase after fertilization, followed by fluctuations throughout development with a considerable decrease at the blastula stage and at the end of gastrulation. Actinomycin has no effect on the activity of thymidylate kinase regardless of when the drug is added to the embryo suspension. Possible regulatory mechanisms of DNA synthesis in sea urchin embryos are discussed: The presence in the unfertilized egg of the most important enzymes controlling the cellular flow of DNA precursors and the availability of dTTP suggest that the block in DNA synthesis observed in the unfertilized egg is due to some particular mechanism that is switched on at fertilization.     

Molting hormone titer changes and their significance during development of the colorado beetle, Leptinotarsa decemlineata

Molting hormone (MH) titer in whole animal extracts of Leptinotarsa decemlineata was determined by chemical extraction and the Musca test (1 MU = 3.5 ng ecdysterone) during the developmental span from newly-ecdysed fourth instar larva to an adult 3 days after eclosion. Within the 17-day period, 21 age groups were chosen to estimate the MH titer. Two peaks of MH titer were detected, one in the post-feeding larval stage and the other during the pupal and pharate adult stage. MH activity was first detected in 2-day-old post-feeding larvae, and reached a maximum of 23.5 MU/g tissue on the third day. It began to decline on 3.5 days, and fell to 5.5 MU/g tissue on 4.5 days, the time of larval-pupal ecdysis. In the pupal and pharate adult stage MH rose after the first day and increased to a maximum of 91.5 MU/g tissue on the third day. The titer again declined on the fourth day, and became undetectable one day before adult emergence and in adults 3 days after emergence. MH was demonstrated to be produced by isolated larval abdomens. A peak of 11.5 MU/g tissue was detected in 7-day post-ligation preparations. The titer decreased to 6.9 MU/g tissue in 10-day post-ligation preparations, which was the time of the ecdysis. The finding raises questions concerning the rôle of MH synthesis by other tissues in relation to the function of the prothoracic glands during insect development.     

Juvenile hormone acid synthesis and HMG-CoA reductase activity in corpora allata of Manduca sexta prepupae

Corpora allata (CA) of last instar larvae of Manduca sexta switch from juvenile hormone (JH) to JH acid secretion just before the onset of wandering behavior. JH acid secretion peaked during the prepupal period and ceased prior to pupal ecdysis. HMG-CoA reductase activity also peaked during the prepupal period and then declined. However, substantial enzyme activity was present in pupal and pharate adult glands. Removal of the brain at the wandering stage caused a reduction in JH acid secretion by prepupal CA. The profile of HMG-CoA activity in CA of debrained larvae resembled that of sham-operated larvae except that the prepupal peak was smaller than in control larvae. Addition of brain extracts to CA maintained in vitro neither stimulated not inhibited JH acid secretion and HMG-CoA reductase activity. It is suggested that the brain regulates CA activity in post-wandering stages via intact nerves.     

Rôle des organes perisympathiques et dynamique de la secretion de la bursicon chez Tenebrio molitor

Light and electron microscope observations have been made on the perisympathetic organs (P.O.) situated on the dorsal somatic abdominal nerves in pupae of Tenebrio molitor.Tests carried out with extracts of P.O. using a bioassay method on pupae of Tenebrio at various developmental stages indicate that these organs have an important role in the storage and release of the tanning hormone, bursicon. The P.O. extracts from the abdominal segments (2 to 5) of 5-day-old pupae were found effective but not those from pharate adult (2 days before ecdysis). The storage and release of bursicon occur later from the P.O. of the posterior abdominal segments (6 to 8).The activity of the blood begins to be effective in the pharate adult 2 days before ecdysis, reaches a maximum 1 hr after the adult ecdysis, and decreases thereafter.This study carried out on the various parts of the nervous system shows that all these parts are effective at a certain stage of the pupal and adult development following an antero-posterior gradient. In the cephalic region, this activity is early and long-lasting, but it appears at later stages and only for a short time in the thorax and abdomen. This phenomenon indicates that bursicon is either passed along the ventral nervous chain or it is synthesized in different parts of the nervous system at successive developmental stages. In the present paper, we have tried to give a comprehensive summary of the dynamics of bursicon in insects.     

Activity of enzymes of arginine metabolism in the cotyledons of developing and germinating pea seeds

Ornithine carbamoyltransferase, argininosuccinate synthetase, argininosuccinate lyase, and arginase activity were measured in extracts from cotyledons of developing and germinating seeds of Pisum sativum L. The course of activity of these four urea cycle enzymes showed a similar pattern during seed development. The activity per cotyledon increased sharply initially and reached a maximum about 5 weeks after anthesis, when the relative water content of the seeds was about 60%. About 8 weeks after anthesis, the seeds were mature (air-dry) and had enzyme activities which were much lower. The activities of the enzymes differed considerably. Ornithine carbamoyltransferase showed the highest activity, followed in order of decreasing activity by arginase, argininosuccinate lyase, and finally argininosuccinate synthetase.

The course of the activity of the four enzymes was different during germination. Arginase activity increased sharply 7 hours after the onset of germination and remained at a constant level during the following days. Argininosuccinate synthetase activity decreased; the other enzymes showed a small increase in activity and a subsequent decrease. Results are discussed in relation to the regulation of the arginine metabolism during pea seed development and germination.

     

Comparative studies of oxidative enzyme systems in epidermis and fat body of diapausing and non-diapausing silkmoths

The activities of four oxidative enzyme systems, including NADH oxidase, succinate-cytochrome c reductase, NADH-cytochrome c reductase, and cytochrome c oxidase, were compared in mitochondrial-microsomal preparations from wing epidermis and fat body of diapausing Samia cynthia pupae, presumptively non-diapausing S. cynthia ricini pupae which were caused to diapause by removal of the brain, and non-diapausing S. cynthia ricini during the pupal and pharate adult period. In diapausing pupae the activities of all enzyme systems were low and presented a profile similar to that previously reported for the Cecropia silkmoth. By contrast, in non-diapausing individuals the activities showed substantially higher levels, and an essentially unchanging pattern from just after the larval-pupal ecdysis through most of adult development. These events are functionally correlated with the patterns of biosynthetic activity in diapausing and non-diapausing silkmoths and are discussed in relation to the endocrine control of diapause and development.     

Haemolymph ecdysteroid titre and epidermal cell commitment of the female southwestern corn borer larvae

The epidermal cell commitment (to pupation or formation of immaculate larvae) and related haemolymph ecdysteroid titres of the southwestern corn borer, Diatraea grandiosella were studied in both nondiapause-bound and diapause-bound last-instar female larvae. Cell commitment was estimated by examining the characteristics of new cuticle secreted in response to an injection of 20-hydroxyecdysone. Haemolymph ecdysteroid titres were determined by radioimmunoassay. Juvenile hormone effect on epidermal cell commitment was studied by applying a juvenile hormone mimic (ZR-515) to last-instar non-diapause-bound larvae and examining the resulting cuticle.In non-diapause-bound larvae, the epidermis of different body regions was committed to pupal development at different times. When pupal cuticular characteristics were evaluated by a scoring system, it appeared that the development of normal pupal cuticle is discontinuous. Three sudden increases in pupal characteristics were observed at 1.67, 2.67 and 3.67 days into the last-larval instar. Haemolymph ecdysteroid titre changes were correlated with the sudden increases in pupal characteristics. Peak ecdysteroid titres were found at 1.67, 2.33, and 3.33 days into the final instar. A fourth ecdysteroid peak (138.8 ng/ml of haemolymph) occurred in pharate pupae. In contrast, the commitment of diapause-bound larvae to produce immaculate integument was made in a fast and continuous fashion. Full commitment was made by 50% of the individuals 4 days (ca. first quarter) into the stadium. Haemolymph ecdysteroid titres fluctuated during the first 2 weeks of the stadium but no significant peaks were observed prior to pharate stage. An ecdysteroid peak (29.8 ng/ml of haemolymph) was identified in pharate immaculate larvae.Pupal development could be completely prevented in 26.7% of nondiapause-bound larvae as late as 4 days into the last instar by topical application of ZR-515. This indicates that the commitment to pupation as revealed by 20-hydroxyecdysone injection is reversible.     

Similarities in properties and a functional difference in purified leucyl-tRNA synthetase isolated from two developmental stages of Tenebrio molitor

In Tenebrio molitor, as well as in other biological systems, there are indications that differences in leucyl-tRNA synthetase activity may play a role in translational control. However, it has not been clear whether the difference in activity is due to the appearance of a multiplicity of enzymes during development or to the alteration of a single enzyme.The purification of leucyl-tRNA synthetase from day 1 and day 7 after the larval pupal molt of Tenebrio molitor is described. The enzyme from both developmental stages was purified over a 1000-fold. The two enzyme preparations are identical in molecular weight (99,000). They show the same characteristics after aging. The pH optimum, heat inactivation behavior, and dependency on divalent cations are the same for both enzymes. They also show identical kinetics with similar values of Km for leucine, ATP, Mg²⁺, and tRNA day 1. However, leucyl-tRNA synthetase purified from day 7 exhibits an additional function in recognizing a new species of isoaccepting tRNA in day 7 tRNA. We have tentatively concluded that the two enzymes are probably different forms of the same enzyme and the additional activity is due to alteration of the enzyme at the macromolecular level during development.     

Thymidine-requiring mutants of Dictyostelium discoideum.

Two thymidine auxotrophs of Dictyostelium discoideum were isolated which improve the efficiency of in vivo DNA-specific radiolabeling. Mutant HPS400 lacked detectable thymidylate synthetase activity, required 50 micrograms of thymidine per ml, and incorporated sixfold more [3H]thymidine into nuclear DNA than did a wild-type strain. Either dTMP or exogenously provided DNA also permitted growth of this strain. The second mutant, HPS401, was isolated from HPS400 and also lacked thymidylate synthetase activity, but required only 4 micrograms of thymidine per ml for normal growth and incorporated 55 times more thymidine label than did a control strain. Incorporation of the thymidine analog 5'-bromodeoxyuridine was also markedly increased in the mutants. Catalytic properties of the thymidylate synthetase of D. discoideum investigated in cell extracts were consistent with those observed for this enzyme in other organisms. These strains should facilitate studies of DNA replication and repair in D. discoideum which require short-term labeling, DNA of high specific activity, or elevated levels of substitution in DNA by thymidine analogs.     

Disk electrophoresis fractionation of hemolymph proteins during the metamorphosis of different castes and sexes of Formica pratensis

Using polyacrylamide electrophoresis the proteins of the haemolymph of the different developmental stages can be separated into eight strong and nine weak coloured fractions during the cocoon period of Formica pratensis. The proteins were stained with aniline black and measured quantitatively by a Chromoscan densitometer. The values were compared with those maintained with bovine serum albumin.The total protein content of the haemolymph was calculated as the sum of the different fractions; at maximum it amounts to 2·1 per cent (w/v). The maximum is reached during the pharate pupal stage and during the pigmentation of the eyes; the minimum can be observed at the end of pupal ecdysis. At the beginning of body pigmentation in all the forms the protein content of the haemolymph was very much reduced, especially in workers and females.All fractions change independently resulting in a different composition of the haemolymph proteins in pharate pupae, eclosed pupae, and pharate adults. The slow-running fractions f1, f3, f5, and f6 and the mean bands f8 and f11 are reduced weakly until body pigmentation, and from the eleventh day strongly in both castes. All fractions are reduced during the cocoon period, but mostly the slow-running ones. Only the front band f14 increases to nearly twice that of the protein content. The importance of the changes in the protein fractions for development of different organs and for the synthesis of the haemolymph proteins and the influence of hormones are discussed.     

Genetic and Immunological Studies of Bacteriophage T4 Thymidylate Synthetase

Thymidylate synthetase, which appears after infection of Escherichia coli with bacteriophage T4, has been partially purified. The phage enzyme is immunologically distinct from the host enzyme and has a molecular weight of 50,000 in comparison to 68,000 for the host enzyme. A system has been developed to characterize T4 td mutants previously known to have impaired expression of phage thymidylate synthetase. For this system, an E. coli host lacking thymidylate synthetase was isolated. Known genetic suppressors were transduced into this host. The resulting isogenic hosts were infected with phage T4 td mutants. The specific activities and amounts of cross-reacting material induced by several different types of phage mutants under conditions of suppression or non-suppression have been examined. The results show that the phage carries the structural gene specifying the thymidylate synthetase which appears after phage infection, and that the combination of plaque morphology, enzyme activity assays, and an assay for immunologically cross-reacting material provides a means for identifying true amber mutants of the phage gene.     

Developmental changes in midgut trehalase activity and its localization in the silkworm, Bombyx mori

The changes in trehalase activity and its localization in the midgut of the silkworm, Bombyx mori, were studied during larval-pupal-adult development. Trehalase activity in larval midgut epithelium increased with the larval growth, reached a maximum level at the middle of the fifth instar, and then decreased gradually. Trehalase activity in larval midgut was found in the epithelial tissue but not in the digestive juice or the midgut contents.The trehalase activity in the whole midgut started to rise at the onset of spinning and increased abruptly at larval-pupal ecdysis to reach an extremely high level 3 days later. This high activity was maintained throughout the subsequent pharate adult development and dropped suddenly at emergence. The midgut trehalase activity during pupal-adult development was mainly found in the midgut contents but scarcely any in the epithelium.Subcellular distribution of midgut trehalase depended upon larval-pupal-adult development. The activity was concentrated in a precipitate fraction of the epithelium until the middle of the fifth instar. During larval-pupal development, however, the activity increased in the soluble fraction with a concomitant decrease in the precipitate fraction. Almost all the trehalase activity in pupal and pharate adult midgut was recovered in the soluble fraction of the midgut contents. The data are discussed from a viewpoint of the histolysis.