POLYAMINE CHANGES DURING DEVELOPMENT OF <i>Drosophila Melanogaster</i>*

Dion, Arnold S.; Herbst, Edward J.

doi:10.1111/j.1749-6632.1970.tb39384.x

Cited by 96 publications

(25 citation statements)

References 18 publications

(1 reference statement)

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“…Putrescine levels rose and then fell during the first 20 hr after transfer to embryogenic and nonembryogenic media. Spermidine levels rose slightly during the time course, whereas spermine levels remained quite constant for the first 6 (3.4 nmol) was supplied to nonembryogenic carrot cells (200 mg fresh weight). Radioactivity was followed as it appeared in putrescine, spermidine, and spermine.…”

Section: Resultsmentioning

confidence: 99%

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Polyamine Metabolism in Embryogenic Cells of Daucus carota

Montague¹,

Koppenbrink²,

Jaworski³

1978

Plant Physiol.

129

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Changes in the metabolsm of polyamines, which seem to be involved in transcription and translation in animal systems, have been studied in cultured cells of Daucus carota (carrot) undergoing embryogenesis. Putrescine levels were elevated by as much as 2-fold over the control within 24 hours after transfer of the celis to embryogenic medium. Spermidine levels were elevated also but spermine levels appeared to be lower in embryogenic celis. Embryogenic celis incorporated ["4Clarginine into putrescine at two times the rate of control celis. These changes suggest that polyamines may be involved in celiular differentiation during embryogenesis.The metabolism of the polyamines, putrescine, spermidine, and spermine, has been the subject of considerable investigation, especially in animal systems (2). Elevated levels of at least one of these compounds have been noted in developing rat fetuses (15), in sea urchin embryos (11), following partial hepatectomy (14), and in transformed cells (3). In addition, there is evidence that polyamines are involved in the regulation of transcription (9,13) and translation (8,10 (5, 16). After addition of an equal volume of 10%1o (w/v) trichloroacetic acid, the homogenates were heated at 80 C for 10 min. The cooled mixture was then centrifuged at 20,000g for 20 min and the supernatant retained for further extraction. After noting the volume of supernatant, a l-ml aliquot was withdrawn for the dansylation procedures (3, 6) used to quantitate the polyamines.A 0.2-ml volume of the supernatant obtained after deproteination corresponding to approximately 7 mg dry cell weight was sufficient for densylation. After making this volume ofsupernatant basic (pH greater than 9) with 18.5 mg of anhydrous Na2CO3, 0.4 ml of dansyl chloride in acetone (30 mg/ml) was added. The stoppered tubes were incubated overnight at 27 C. The excess dansyl chloride was destroyed by the addition of 0.1 ml of a proline solution (100 mg/ml in H20) and the dansylated products were extracted into 0.5 ml of benzene. Aliquots of 10 to 15 [LI of the benzene extract were supplied to activated (45 min at 100 C) silica gel TLC plates. The plates were developed for 5 hr in ethyl acetate-cyclohexane (2:3, v/v) and immediately sprayed with 10% (v/v) triethanolamine in isopropyl alcohol. The polyamines were quantified with a Turner model III fluorometer equipped with a Camag T Scanner Mark III (activation at 365 mm) and peak areas (>512 nm) from the extracts were compared to those obtained from dansylation of standards. It was necessary to include at least two standard spots on each plate along with the extract samples because there was a variation of fluorescence both between plates and upon a single plate.In order to determine radioactivity present in the polyamines after exposure to ["Clarginine, 2.5 ml of the supernatant obtained after deproteination were extracted four times with 2 volumes of ether to remove the trichloroacetic acid. Then, the extract was made basic by the addition of 0.05 ml of 10 N NaOH per ml an...

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Section: Resultsmentioning

confidence: 99%

“…The cooled mixture was then centrifuged at 20,000g for 20 min and the supernatant retained for further extraction. After noting the volume of supernatant, a l-ml aliquot was withdrawn for the dansylation procedures (3,6) used to quantitate the polyamines.…”

mentioning

confidence: 99%

Polyamine Metabolism in Embryogenic Cells of Daucus carota

Montague¹,

Koppenbrink²,

Jaworski³

1978

Plant Physiol.

129

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“…For determinations of polyamine content of cells and media, Millipore filters containing the filtered cells were extracted in 1.0 ml of 0.2 N perchloric acid. To the filtrate (medium) was added 2.0 N perchloric acid to a final concentration of 0.2 N. The acid extracts were dansylated as described (10).…”

Section: Methodsmentioning

confidence: 99%

Polyamine Stimulation of Nucleic Acid Synthesis in an Uninfected and Phage-Infected Polyamine Auxotroph of Escherichia coli K12

Dion

Cohen

1972

Proc. Natl. Acad. Sci. U.S.A.

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The addition of arginine to cultures of Escherichia coli K12 deficient in agmatine ureohydrolase (EC 3.5.3 Two constitutive pathways function in the synthesis of putrescine in Escherichia coli (1, 2). In the E. coli K12 mutant deficient in agmatine ureohydrolase (EC 3.5.3.7) (3), the conversion of agmatine to putrescine is blocked. The addition of arginine prevents putrescine formation in this mutant by inhibition of the synthesis of ornithine from glutamate. Such a polyamine auxotroph has permitted us to investigate the dependence on polyamines of RNA and DNA synthesis in uninfected cells and the possible requirement for polyamines for DNA synthesis in phage-infected cells.In 1957, Hershey (4) described two low molecular weight ninhydrin-positive compounds in purified T-even phage preparations; these amines were also demonstrated to be present in the uninfected host. These compounds were shown to be derived from arginine, and were concomitantly injected with phage DNA upon infection; they were identified as putrescine and spermidine by Ames and coworkers (5, 6), who showed that the polyamine content of the T-even phage could neutralize 40-50% of the DNA phosphates. Cohen and Raina (7) have demonstrated that the net synthesis of polyamines after infection parallels DNA synthesis. The MATERIALS AND METHODSSome properties of E. coli K12 (MA159) (His-, Leu-, Thr-) defective in agmatine ureohydrolase activity have been described (3). The characterization of bacteria bearing this block has been described (3, 9). The mutant that was used in this study, MA159, was kindly given to us by Dr. W. Maas. The organism is derived from a thy-derivative (MA145) of MA135 (3) by transduction into a ser A strain, PA260. In this study, two methods of polyamine depletion were used. In method 1 (J. Poindexter, personal communication), an overnight broth culture is chilled 5 hr at 4°C and diluted 1: 20 in AFA medium (AF medium, see ref. 3, supplemented with 100 ,ug of arginine/ml). 0.1-ml aliquots are plated on AFA agar plates, and after overnight growth at 37°C, bacteria are scraped from the agar surface with small aliquots of AFA medium. These harvested bacteria are then centrifuged at 4080 X g for 15 min, and resuspended in Davis-Mingioli medium (9) without glucose. After an additional wash, aliquots of the suspension are added to various AF media containing the designated supplements.In method 2, an overnight broth culture is chilled 5 hr at 4°C and diluted 1:40 in AFA medium (final concentration of arginine = 100 ,g/ml). Overnight growth at 37°C results in polyamine-depleted cells. These are centrifuged and washed in Davis-Mingioli salts medium without glucose. Aliquots of a 4-fold concentrated suspension are added to AF media containing the designated supplements.Lysates of T4r+D were prepared by infection of E. coli B at a multiplicity of infection (MOI) of 0.1 and incubation overnight. Purified phage stocks were obtained by two cycles of low (4080 X g)-and high (27,300 X g)-speed centrifugation of overnight brot...

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“…Methods For polyamine analysis of bacterial extracts, cells were harvested and washed at room temperature with buffer A (Tris/HCl, pH 7.8, 0.05 M; NH4Cl, 0.06 M; magnesium acetate, 5 mM; mercaptoethanol, 6 mM). Bacterial pellets were then extracted with 0.2 M-HCl04 at 4°C and, after centrifugation at 10000 g for 10 min, the supernatant fluids were dansylated as described by Dion & Herbst (1970). The resulting dansyl derivatives as well as those of standard polyamine solutions were extracted with toluene and analysed by t.l.c.…”

Section: Experimental Materialsmentioning

confidence: 99%

A probable new pathway for the biosynthesis of putrescine in Escherichia coli

Cataldi¹,

Algranati²

1986

Biochemical Journal

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Some cultures of Escherichia coli BGA8, a mutant unable to synthesize putrescine, showed a change of behaviour and could grow almost equally well in either the absence or the presence of polyamines after repeated periods of polyamine starvation. Experiments in vivo with radioactive precursors showed that the bacteria which evaded the polyamine requirement had recovered their ability to synthesize putrescine from glucose or glutamic acid, but not from ornithine or arginine. These results are in agreement with the fact that the polyamine-independent cells were still deficient in the enzymes ornithine decarboxylase and agmatinase. Our findings seem to indicate the existence of a new pathway to synthesize putrescine which does not involve ornithine or arginine as intermediates.

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POLYAMINE CHANGES DURING DEVELOPMENT OF *Drosophila Melanogaster**

Cited by 96 publications

References 18 publications

Polyamine Metabolism in Embryogenic Cells of Daucus carota

Polyamine Metabolism in Embryogenic Cells of Daucus carota

Polyamine Stimulation of Nucleic Acid Synthesis in an Uninfected and Phage-Infected Polyamine Auxotroph of Escherichia coli K12

A probable new pathway for the biosynthesis of putrescine in Escherichia coli

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POLYAMINE CHANGES DURING DEVELOPMENT OF Drosophila Melanogaster*

Cited by 96 publications

References 18 publications

Polyamine Metabolism in Embryogenic Cells of Daucus carota

Polyamine Metabolism in Embryogenic Cells of Daucus carota

Polyamine Stimulation of Nucleic Acid Synthesis in an Uninfected and Phage-Infected Polyamine Auxotroph of Escherichia coli K12

A probable new pathway for the biosynthesis of putrescine in Escherichia coli

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POLYAMINE CHANGES DURING DEVELOPMENT OF *Drosophila Melanogaster**