Embryo Cytoplasmic extract

Fig. 12. Informosomes from loach embryo extracts labeled for 3 hours with uridine. Dotted lines, radioactivity solid lines, optical density. A. Sucrose density gradient centrifugation of cytoplasmic extract (22,000 rpm for 18 hours). B. Recentrifugation of the material of 73 to 62S peak in CsCI density gradient. C. The same but of 45 to 36S zone. (From Ovchinnikov et al. 1960a. Molec. Biot. (U.S.S.RJ, 3 448 3.)...

Protocol 33.1 Embryo Collection and Dechorionation, 571 Protocol 33.2 Preparation of the Cytoplasmic Extract, 573... 

Preparing Cytoplasmic Extracts from Drosophila Embryos ... 

The production of a high-quahty Drosophila embryonic cytoplasmic extract for use in protein purification or biochemistry is relatively easy, providing population cages are available that produce at least 5 g of embryos during a 3-hour period (for methods to maintain population cages, see Sisson, this volume). Smaller quantities of embryos can be used to produce extracts that are useftil for in vitro biochemical assays (see, e.g., Moritz et al. 1998), and thus it should be possible to make extracts from mutant stocks that could then be tested in vitro. Protocol 33.1 describes the collection and dechorionation of embryos for making cytoplasmic extracts. It is fairly easy to prepare even very concentrated cytoplasmic extracts from Drosophila embryos, with protein concentrations of 50-75 mg/ml (see Protocol 33.2). 

The DNA is certainly not identical with the cytoplasmic inhibitor which was extracted from the 105,000 g supernatant of chick embryos. The partially purified cytoplasmic inhibitor does not contain any DNA and the inhibitory activity is not destroyed by DNase, whereas the inhibition by DNA is completely abolished after treatment with DNase (Tiede-mann et al., 1972a). 

This protocol is an adaptation of Murray (39). Briefly, a crude cjdostatic factor (CSF) arrested egg extract (cytoplasm arrested in meiotic metaphase) is prepared. Calcium is then added to allow the extract to progress into interphase, and a high-speed spin is performed to obtain a purer cjdoplasmic fraction. Cytochalasin is omitted from the protocol, since the carryover of cytochala-sin into the final extract used for sperm incubations interferes with the normal development of transplant embryos. Use of high-speed rather than crude... 

The amount of jSRNA began to increase in the early blastula stage which correlated with the accumulation of the cytoplasmic mRNA which contained poly (A). The maximum amount of aRNA (25% of the total heterogenous nuclear RNA) was obtained in the middle blastula stage (Dubroff and Nemer, 1975/76). The ratio of mRNA s with and without poly (A) sequences correlated with the increasing of size of free polysomes in the sea urchin embryo (Nemer and Surrey, 1975/76 Nemer et al., 1975). It is possible to translate different mRNA fractions extracted from sea urchin and amphibian embryos in the cell-free system (Ruder-man and Pardu, 1977). 

A cytoplasmic factor is postulated to be responsible for the initiation of the S phase, and various investigators have demonstrated that DNA synthesis can be initiated by extracts of He La cells (Friedman and Mueller, 1968 Kumar and Friedman, 1972), L cells and ascites cells (Thompson and McCarthy, 1968), and embryos and eggs of Xenopus (Benbow and Ford, 1975). Although these factors are thought to be proteins, their exact nature and mechanisms of action are unknown. Recently it was suggested that diadenosine-tetra-phosphate (Ap4A) may act as an initiator of DNA replication in animal cells (Grummt, 1978). 

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