Abstract
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.
Highlights
5-Bromo-2’-deoxyuridine (BrdUrd), an analogue of thymidine, is normally incorporated into DNA as 5-bromouracil in place of thymine
We show that if BrdUrd is injected at a concentration of 8 mM or less into larvae of B. hygida during the period of gene amplification, adults are produced at the same frequency as when larvae are injected with water
If larvae at age E3 are injected with 1 μL of a BrdUrd solution at a concentration of 8 mM or less, they will develop into healthy adult flies, in numbers similar to those produced from larvae treated with water, without a significant delay in development
Summary
5-Bromo-2’-deoxyuridine (BrdUrd), an analogue of thymidine (dThd), is normally incorporated into DNA as 5-bromouracil in place of thymine. In Bradysia hygida, the process of gene amplification starts at the same time at all chromosome sites (age E3) that later will form DNA puffs, in the anterior (S1) and posterior (S3) regions of the salivary gland. RNA synthesis is hindered at chromosomal sites that would normally form DNA puffs These effects appear to be a consequence of BrdUrd incorporation into the amplified DNA. The present results indicate that in some instances, in other systems, the inhibitory effects of BrdUrd on the activity of specific genes can be due to a localized and irreversible chromatin compaction. These morphological observations are the basis for a molecular investigation that is presently underway
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