Separation of Drosophila RNAs on acrylamide gels in formamide

Mitchell, Herschel K.; Chen, Pei-Shen; Lipps, Loveriza S.; Moller, Galina

doi:10.1016/0020-1790(78)90007-0

“…From 10or 11-day-old virgin male flies, male accessory glands were dissected free of testicular and intestinal (gut) tissue and stored in a drop of ice-cold MOPS buffer [10 mM MOPS (4-morpholinepropanesulfonic acid, Sigma), pH 7.0, 80 mM NaCl, 10 mM KC1, 0.2 mM MgCl2, and 0.1 mM CaCl2 (Mitchell et al, 1978)]. The glands from six flies were rinsed and then incubated at room temperature for 1 h with gentle shaking in 1 -mL homogenization tubes with 60 mL of MOPS buffer containing variable concentrations of CaCl2, 2 pCi of [35S] methionine [New England Nuclear (1125 Ci/mmol)] or 25 pCi of [32P] orthophosphate [Amersham (1000 Ci/mmol)],…”

Section: Methodsmentioning

confidence: 99%

Ribosome subunit to polysome ratios affect the synthesis of rRNA in Drosophila cells

Yamamoto

¹

,

Pellegrini²

1990

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Many investigations have revealed that ribosome numbers increase in parallel with the growth rate of cells. Here we show that the absolute level of protein synthesis may not be the only factor influencing rRNA synthesis in a nondividing eukaryotic cell. Under conditions of complete (greater than 99%) inhibition of protein synthesis by four different antibiotics, there is a corresponding inhibition of rRNA synthesis. At lower levels of inhibition of protein synthesis (70%), a different effect of individual antibiotics on rRNA synthesis is observed. Cycloheximide and anisomycin, which cause a decrease in the free subunit pool due to a buildup of polysomes, stimulate rRNA synthesis, whereas puromycin and pactamycin, which cause an increase in the free subunit pool, cause a decrease in rRNA synthesis. These effects on rRNA synthesis are not solely due to a low level of completed proteins. Pactamycin treatment allows completed proteins to be made yet lowers rRNA labeling, while anisomycin treatment does not show synthesis of complete proteins yet increases rRNA labeling. The result suggest that eukaryotic cells may regulate ribosome synthesis in response to the number of free versus translating (polysomal) ribosomes as do Escherichia coli cells.

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“…For labeling in culture, dissected salivary gland lobes were incubated in a 5-to 10-lA drop of culture medium containing 10 pCi of a radiolabeled amino acid per ,ul (21,29 3.4 p.Ci/cm2. Larvae were then transferred to fresh filter paper and left there until pupation.…”

Section: Methodsmentioning

confidence: 99%

The structural genes for three Drosophila glue proteins reside at a single polytene chromosome puff locus.

Crowley¹,

Bond²,

Meyerowitz³

1983

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The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7.Drosophila melanogaster begins life as a fertilized egg, which after a day of embryonic development hatches as a wormlike first-instar larva. After another day this larva molts and becomes a larger, second-instar larva; one further day leads to a second molt and the last stage of larval life, the third larval instar. This lasts two days, at the end of which the larval cuticle hardens to form a pupal case. After an immobile prepupal period, pupation and metamorphosis occur, resulting in an adult fly. The major synthetic activity of the salivary gland cells of the third-instar larva is the production of a set of about 10 polypeptides that are secreted into the lumen of the gland after synthesis and that at the end of larval life are expelled through the duct of the salivary glands and deposited on the surface upon which the larva rests. This protein secretion serves as a glue that causes the prepupa, and subsequently the pupa, to adhere to its substrate for the duration of the pupal period (5,8,15,16

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“…Salivary gland lobes were then dissected from the larvae, and proteins were extracted as described below. For labeling in culture, dissected salivary gland lobes were incubated in a 5to 10-lA drop of culture medium containing 10 pCi of a radiolabeled amino acid per ,ul (21,29 [3H]threonine, 5.3. After 30 min at 22 to 24°C the glands were removed from the labeling medium, and proteins were extracted from salivary gland lobes by one of the following four procedures.…”

Section: Methodsmentioning

confidence: 99%

The Structural Genes for Three Drosophila Glue Proteins Reside at a Single Polytene Chromosome Puff Locus

Crowley

¹

,

Bond

²

,

Meyerowitz

³

1983

Molecular and Cellular Biology

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The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7.

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Separation of Drosophila RNAs on acrylamide gels in formamide

Cited by 13 publications

References 11 publications

Ribosome subunit to polysome ratios affect the synthesis of rRNA in Drosophila cells

Ribosome subunit to polysome ratios affect the synthesis of rRNA in Drosophila cells

The structural genes for three Drosophila glue proteins reside at a single polytene chromosome puff locus.

The Structural Genes for Three Drosophila Glue Proteins Reside at a Single Polytene Chromosome Puff Locus

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