Steve Miller

The essential role of folic acid and the effect of antimetabolites on growth and metamorphosis of housefly larvae Musca domestica L.

Abstract Housefly larvae reared on a defined diet under aseptic conditions show complete dependence on dietary folic acid (PGA) for normal growth and metamorphosis. Larvae cannot synthesize PGA from available precursors; neither can they synthesize DNA and RNA from purine and pyrimidine bases in the absence of folic acid. Aminopterin and pyrimethamine reversibly inhibit the enzyme folic acid reductase which catalyses the reduction of folic acid to tetrahydrofolic acid. As a consequence, larval growth and metamorphosis are impaired. 5-Fluorouracil, azaserine, 3-aminotriazole and fluoroacetamide are potent inhibitors of larval development but their action is not reversed by folic acid.

Metabolic purine pathways in the developing ovary of the housefly, Musca domestica

Abstract 1. 1. A detailed knowledge of the enzymatic pathways of purine biosynthesis and interconversions in houseflies is important in determining if any biochemical differences exist that might allow exploitation in the selection of compounds that can be used in insect control. 2. 2. Accordingly, the present study demonstrates that the housefly ovaries are deficient in hypoxanthine-guanine phosphoribosyltransferase. When (8- 14 C) adenine was injected into houseflies prior to sacrifice, the purine was utilized in the formation of ovarian DNA adenine and guanine. 3. 3. The transformation of adenine into guanine is not reversible since no activity was observed in the adenine fraction when radioactive guanine was administered. Adenine phosphoribosyltransferase appears to be the main salvage pathway enzyme of nucleotide synthesis. 4. 4. There was no evidence to indicate the presence of purine nucleoside phosphorylase or guanosine monophosphate reductase. 5. 5. The nucleoside kinases were present since radioactive guanosine was incorporated into guanine of DNA. The animal and bacterial cells have low kinase activity. 6. 6. The data indicate that free purine base analogues are not inhibitors of nucleic acid synthesis and nucleotide formation is necessary for inhibition in the salvage pathway.

RNA synthesis in the developing ovary of the housefly: Incorporation of 14C-formate

Abstract RNA synthesis in the developing ovary of the housefly was studied by determining nucleic acid concentration and isotopic incorporation into the purines of RNA. A method is described for the isolation and purification of cytoplasmic RNA. The base composition of the RNA was DNA-like, with a low (G+C) (A+U) ratio. Administration of 14C-labelled formate to female houseflies leads to extensive incorporation of 14C into adenine and guanine moieties of RNA in the early stages of ovarian development. Incorporation of the isotope into ovarian RNA reached a maximum at 12 hr after its administration.

DNA synthesis in the developing ovary of the housefly

Abstract 1. 1. A method is described for the isolation of DNA from housefly ovaries where small quantities of the nucleic acid are present. 2. 2. Contamination of DNA by residual protein was reduced by treatment with proase. All the DNA samples were essentially free of RNA, as indicated by the orcinol reaction. 3. 3. The average molar base composition (40 per cent of guanine+cytosine) corresponds to Drosophila DNA, and the purine to pyrimidine ratio is close to unity, as would be expected for double stranded DNA. 4. 4. The conversion of (2- 14 C) glycine to purines of DNA was used as a measure of the de novo biosynthetic route. Analysis of the DNA, after hydrolysis, demonstrated that the label is present in the purine and pyrimidine bases. 5. 5. The specific activity is highest in the bases of DNA extracted from ovaries from 24-hr-old flies. The decrease in specific activity thereafter is due to the diluting effects in the de novo synthesis.

Utilization and interconversion of purines and ribonucleosides in the mosquito Anopheles albimanus Weidemann

1. 1. The salvage pathways of ribonucleotide synthesis in the adult mosquito, Anopheles albimanus, were established by the incorporation of 8-14C-labelled purines and ribonucleosides into the RNAs. 2. 2. A comparison of enzymatic activities shows that adenine phosphoribosyltransferase is the most active enzyme in purine nucleotide synthesis. 3. 3. Adenosine is presumably deaminated to inosine. 4. Evidence is presented for the existence of inosine kinase. 4. 5. The data show low hypoxanthine-guanine phosphoribosyltransferase activity. For comparison, the activity of this enzyme is considered in the housefly, Mucosa domestica. 5. 6. The transformation of adenine into guanine is reversible, due to detectable activity found in RNA adenine when radioactive guanine was the precursor. 6. 7. This finding establishes the presence of guanosine monophosphate reductase 7. 8. From the results, it was concluded that adenine phosphoribosyltranferase is a metabolically important enzyme in mosquito maturation and that inhibitors of the enzyme may have potential value as insect control agents.

One-carbon metabolism in the developing ovary of the housefly Musca domestica

Abstract Adult female houseflies incorporate one-carbon units from radioactive formate, glycine, and serine into the purines of ovarian RNA. When RNA was extracted from the ovaries and hydrolysed, the purine bases were labelled in a manner suggesting that radioactive substrates were incorporated into RNA by recognized biosynthetic pathways involving a one-carbon pool. The incorporation reached a maximum value in 12-hr-old flies with formate as substrate and in 48-hr-old flies with glycine and serine as substrates. The metabolism of (2- 14 C) glycine and (3- 14 C) serine has been compared with that of ( 14 C) formate. Formate enters the purines to the greatest extent followed by the α-carbon atom of glycine and the β-carbon atom of serine. The concept of active formate has been established in the developing ovary of the housefly.

RNA synthesis in two species of anopheline mosquitoes

Abstract . 1. 1. A method is described for the extraction of adult mosquito nuclear RNA. The RNA is tightly bound to nuclear components. The nuclei were dispersed by the use of sodium chloride and urea of high ionic strength. 2. 2. Deproteinization of the cytoplasmic minus mitochondria fraction resulted in the separation of a high molecular weight RNA. The RNA is derived from a polynucleotide precursor in the nucleus and transported to the cytoplasm. 3. 3. The rate of RNA synthesis in Anopheles quadrimaculatus and A. albimanus was measured by the incorporation of radiolabelled adenine into RNA. The radioactive precursor was presumably converted to the adenine nucleotide by adenine phosphoribosyltransferase. 4. 4. The conversion of adenine nucleotides to guanine nucleotides was essentially the same in the two species, however, differences were found in the specific activities of RNA purines. 5. 5. Cellulose powder and DEAE profiles of nuclear and cytoplasmic RNAs from the two species were compared.

The nature of the changes in the pattern of RNA synthesis by the juvenile hormone analogue altosid

Abstract The effect of various concentrations of Altosid and actinomycin D under defined conditions on housefly metamorphosis was investigated with three strains of houseflies. The morphogenetic response varied with the strains and the length of time which the larvae were exposed to the juvenile hormone analogue. De novo RNA synthesis was studied with (2-14C)-glycine. Methods were developed for the isolation of nuclear, soluble, and ribosomal RNA. The procedure presented provides a DEAE-cellulose chromatographic method for the removal of high molecular weight RNA from DEAE at a neutral pH. Labelling of the RNAs was increased in the presence of the juvenile hormone analogue indicating an increase in the rate of RNA synthesis. The higher incorporation of the labelled precursor into nuclear RNA demonstrates that cytoplasmic RNA is derived from the nuclei.

The effect of vinblastine sulphate on the incorporation of [2-14C]glycine into housefly DNA

Abstract The synthesis of DNA was studied in houseflies 48 hours after emergence in the presence and absence of vinblastine sulphate (VLB). In all cases administration of the alkaloid inhibited the incorporation of [2- 14 C]glycine into DNA. The site of inhibition has not been established. Analysis of the bases in DNA revealed that the specific activities of DNA purines were decreased (relative to control values) more than those of DNA thymine. VLB caused sterility in the female flies but not in the male flies. A method is described for preparing a reproducible preparation of high quality DNA from houseflies.

Isolation, purification and characterization of N5-formyltetrahydrofolic acid (folinic acid) from the housefly

Abstract Methods are presented for the isolation and purification of N 5 -formyltetrahydrofolic acid from the housefly. By the complimentary effect of DEAE column chromatography and repeated thin layer chromatography the folate derivative was sufficiently purified to give an ultraviolet spectrum identical with authentic folinic acid. Other characteristics presented are also similar.