RESUMO
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.
Assuntos
Bromodesoxiuridina/farmacologia , DNA/efeitos dos fármacos , Dípteros/genética , Proteínas de Insetos/efeitos dos fármacos , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/efeitos dos fármacos , Animais , Autorradiografia , Diferenciação Celular , Proteínas de Insetos/genética , Larva/efeitos dos fármacos , Glândulas Salivares/efeitos dos fármacos , Proteínas e Peptídeos Salivares/genéticaRESUMO
When the first group of DNA puffs is active in the salivary gland regions S1 and S3 of Bradysia hygida larvae, there is a large increase in the production and secretion of new salivary proteins demonstrable by [3H]-Leu incorporation. The present study shows that protein separation by SDS-PAGE and detection by fluorography demonstrated that polypeptides range in molecular mass from about 23 to 100 kDa. Furthermore, these proteins were synthesized mainly in the S1 and S3 salivary gland regions where the DNA puffs C7,C5,C4 and B10 are conspicuous, while in the S2 region protein synthesis was very low. Others have shown that the extent of amplification for DNA sequences that code for mRNA in the DNA puffs C4 and B10 was about 22 and 10 times, respectively. The present data for this group of DNA puffs are consistent with the proposition that gene amplification is necessary to provide some cells with additional gene copies for the production of massive amounts of proteins within a short period of time (Spradling AC and Mahowald AP (1980) Proccedings of the National Academy of Sciences, USA, 77: 1096-1100).
Assuntos
Animais , Feminino , Dípteros/genética , Sondas de DNA/genética , Amplificação de Genes/genética , Técnicas In Vitro , Peptídeos/biossíntese , Glândulas Salivares/fisiologia , Saliva/química , Eletroforese , Eletroforese em Gel de Poliacrilamida , RadioatividadeRESUMO
A recombinant clone carrying a 2-kb fragment was isolated from a mini-library of the B10 DNA puff of Bradysia hygida. This fragment was amplified in the salivary gland during the period of DNA puff formation. Amplification started when DNA puff anlage was formed and continued to increase, reaching a maximum of abouth 10-fold 28 h later. Northern blot hybridization experiments showed that this 2-kb fragment was complementary to two RNA species of about 1.3 kb and 1.1 kb, which are developmentally regulated in the salivary gland. Maximum amounts of these messages were present when the B10 puff is fully expanded