5-Bromo-2’-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae)

5-Bromo-2’-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment ofBradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

5-Bromo-2'-deoxyuridine induces visible morphological alteration in the DNA puffs of the anterior salivary gland region of Bradysia hygida (Diptera, Sciaridae).

5-Bromo-2'-deoxyuridine (BrdUrd) has long been known to interfere with cell differentiation. We found that treatment of Bradysia hygida larvae with BrdUrd during DNA puff anlage formation in the polytene chromosomes of the salivary gland S1 region noticeably affects anlage morphology. However, it does not affect subsequent metamorphosis to the adult stage. The chromatin of the chromosomal sites that would normally form DNA puffs remains very compact and DNA puff expansion does not occur with administration of 4 to 8 mM BrdUrd. Injection of BrdUrd at different ages provoked a gradient of compaction of the DNA puff chromatin, leading to the formation of very small to almost normal puffs. By immunodetection, we show that the analogue is preferentially incorporated into the DNA puff anlages. When BrdUrd is injected in a mixture with thymidine, it is not incorporated into the DNA, and normal DNA puffs form. Therefore, incorporation of this analogue into the amplified DNA seems to be the cause of this extreme compaction. Autoradiographic experiments and silver grains counting showed that this treatment decreases the efficiency of RNA synthesis at DNA puff anlages.

Acid phosphatase activity in mature secretory granules of the salivary gland of Bradysia hygida.

It is uncommon to find acid phosphatase activity in mature secretory granules. This paper demonstrates by light and electron microscope cytochemistry an acid phosphatase in mature secretory granules in the cells of one region of the salivary gland of Bradysia hygida (Diptera, Sciaridae). These secretory granules increase in number during larval development up to the beginning of the pre-pupal period when they undergo massive exocytosis. Biochemical assays show that upon exocytosis of the majority of the granules the total acid phosphatase activity in the granular gland region drops to 10% of the maximum reached before exocytosis. During and after exocytosis, two other acid phosphatases, eletrophoretically different and much weaker in activity, become increasingly detectable in all gland regions. At the same time, in whole mount preparations, numerous tiny acid phosphatase-positive granules (probably secondary lysosomes) become evident in all major cell types of the salivary gland. These results indicate that the S2 region of the salivary gland has mature secretory granules containing an acid phosphatase destined for exocytosis which is different in molecular properties from other acid phosphatases (likely lysosomal) made by the gland.

The effect of perchloric acid fixation on DNA preservation in the salivary gland of Bradysia hygida.

In Bradysia hygida (Diptera, Sciaridae) well spread polytene chromosomes, free of cytoplasm, and with good morphology are consistently obtained if before squashing in acetic acid solution, the salivary glands are fixed in 7% perchloric acid containing small amounts of ferric ions (SAUAIA et al. 1971). We show here that, with regard to the preservation of total incorporated 3H-thymidine and with regard to the relative autoradiographic labelling of expanded chromosome regions as compared to the labelling of a non-expanded one, this method is equivalent to fixing the salivary glands in ethanol-acetic (3:1). We show also that if this kind of preparation is subject to mild acid hydrolysis a small amount of the total 3H-labelled material may be lost.

Development of DNA puffs and patterns of polypeptide synthesis in the salivary glands of Bradysia hygida.

In the three salivary gland regions of Bradysia hygida (Diptera, Sciaridae) the patterns of polypeptide synthesis, as revealed by electrophoresis and fluorography, are very stable during the fourth larval instar until about 30 h before the pupal molt. At this age the patterns of polypeptide synthesis start to undergo marked changes. The striking correlations between these changes and the development of two distinct groups of DNA puffs support the proposal that DNA puffs are causally related to the synthesis of specific proteins in the salivary glands.

The Feulgen reaction: importance of the wash after hydrolysis.

Washing in absolute ethanol after FEULGEN hydrolysis minimizes losses of DNA. The hydrolysis in HCl diluted in tetrahydrofuran is not superior in this respect to conventional hydrolysis if this is followed by an ethanol wash. These conclusions are based on cytophotometric and radiometric experiments made on salivary glands of sciarid larvae.