Abstract
DNA synthesis has been studied in polytene nuclei isolated from larval salivary glands of Drosophila hydei. The incubation conditions employed promote maximum incorporation of TTP-H3 and retention of normal polytene chromosome morphology. The chromosome structure is sensitive to the Mg2+ concentration; a normal banding pattern is observed between 4 and 10 mM Mg2+. At the optimum pH of 7.8, incorporation continues for over an hour. All four deoxyribonucleoside triphosphates are required for maximum incorporation. The reaction is stimulated by 0.6 mmATP and strongly inhibited at higher ATP concentrations. Competition experiments demonstrate that either TDP or TTP is the effective labeled precursor. The labeled product is sensitive to DNase and has a density identical to that of nuclear DNA. Autoradiographs prepared from spread chromosomes demonstrate that discontinuous and continuous labeling patterns observed in vivo are also produced with isolated nuclei in the absence of cytoplasmic factors. Incubation of the isolated nuclei results in a low level of uniform incorporation that is superimposed on the normal autoradiographic pattern obtained after in vivo labeling. This background incorporation can be greatly increased by prior irradiation of the glands. The presence of exogenous DNA during nuclear incubation stimulates total incorporation. These observations demonstrate that the isolated nuclei possess a reserve synthetic capacity. About 20% of the isolated nuclei are inactive in DNA synthesis.
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This investigation was supported by PHS Research Grant No. 5 R01 GM 16298 from the National Institute of General Medical Sciences.
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Boyd, J.B., Presley, J.M. Deoxyribonucleic acid synthesis in isolated polytene nuclei of Drosophila hydei . Biochem Genet 9, 309–325 (1973). https://doi.org/10.1007/BF00486067
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DOI: https://doi.org/10.1007/BF00486067