L y s + RNA in the sections was followed in situ using autoradiography (fig. 28). Immediately (< 10 min) after injection the RNA is localised in the injected region (fig. 28c and d ) . Although the injected RNA occupies a large volume in the oocyte, it does not appear to have caused any major disruption of the cytoplasm as judged by the pattern of yolk platelets in the section (fig. 28a and b ) . Since serial sections were made of the injected oocytes it was possible to estimate the shape of the region occupied by the injected RNA. In most cases it appeared ellipsoid or ovoid rather than spherical.
After 6h some movement of the RNA had taken place, although most remained localised round the injection site (fig. 28e and f). By 24h the injected RNA had reached all parts of the oocyte. However, although the vegetal pole injected oocyte had an almost even distribution of
Figure 28. Autoradiographs of Injected oocvtes
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F-labelled Lys dimethyl cap RNA was injected into oocytes at the vegetal (a, b, c, e and g) or the animal pole (d, f and h ) , and the oocytes were fixed in boulns fixative within 10 min (a. b, c, d ) , 6 hours (e, f) and 24 hours (g, h) of injection. After fixation the oocytes were dehydrated and embedded in paraffin wax. Serial sections were made of the oocytes and mounted on slides. After removal of the wax the sections were autoradiographed by coating the slides with a liquid photographic emulsion. After development of the autoradiographs, the sections were visualised by phase-contrast (a), or dark-field (b-h) illumination. Panels (a) and (b) show the same section under phase- contrast and dark-field illumination.
Under dark-field illumination (b-h) the silver grains in the
autoradiographs appear as bright sources of light. The bright rim round the oocytes in panels b-h, is due to light scattering by the dense pigment granules (not silver grains) around the periphery of tha oocytes. The nucleus of the oocyte is indicated (g.v., germinal vesicle) . The concentrations of grains in the g.v. of the oocytes in panels (g) and (h) lie over nucleoli. The position of the nucleoli in these sections was found using phase-contrast illumination (data not shown).
Figure 28. Autoradiographs of injected oocytes
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P-labelled Lys dimethyl cap RNA was injected into oocytes at the vegetal (a, b, c, e and g) or the animal pole (d, f and h) , and the oocytes were fixed in bouins fixative within 10 min (a, b, c, d ) , 6 hours (e, f) and 24 hours (g, h) of injection. After fixation the oocytes were dehydrated and embedded in paraffin wax. Serial sections were made of the oocytes and mounted on slides. After removal of the wax the sections were autoradiographed by coating the slides with a liquid photographic emulsion. After development of the autoradiographs, the sections were visualised by phase-contrast (a), or dark-field (b-h) illumination. Panels (a) and (b) show the same section under phase- contrast and dark-field illumination.
Under dark-field illumination (b-h) the silver grains in the
autoradiographs appear as bright sources of light. The bright rim round the oocytes in panels b-h, is due to light scattering by the dense pigment granules (not silver grains) around the periphery of ths oocytes. The nucleus of the oocyte is indicated (g.v., germinal vesicle). The concentrations of grains in the g.v. of the oocytes in panels (g) and (h) lie over nucleoli. The position of the nucleoli in these sections was found using phase-contrast illumination (data not shown).
concentration round the germinal vesicle (fig. 2 8 h ) . This correlates with the concentration of endogenous oocyte RNA in this region (see fig. 9). One major limitation of this method of analysis is that the distribution of grains is caused both by intact injected RNA, partially degraded RNA, single labelled nucleotides and nucleotides that have been reincorporated into endogenous RNA. In particular detectable amounts of ribosomal RNA form (see fig. 17), which accounts for the localisation of grains over the nucleoli found in the autoradiographs of some of the sections. However from the measurement of RNA stability (Table 2) at least 45% of the grains are formed by injected RNA at 24h. Also to confirm that the pattern of grains in the autoradiographs is a genuine reflection of the RNAs distribution, movement was analysed as before. Frozen oocytes were sectioned and the RNA from the animal and vegetal halves was run on gels, which were dried down and autoradiographed (fig. 29). The distribution of RNA was quantified by cutting up and scintillation counting the relevant parts of the gels (Table 5). The distribution of RNA on the gels at 24h agrees with the distribution of grains in the in situ autoradiographs. There is a concentration of RNA in the animal half and movement from the vegetal to the animal half of the oocyte is 1.4 times faster than in the opposite direction. In a separate experiment using a different batch of oocytes movement from the vegetal to animal half was 1.7 times faster than in the opposite
direction.
SLl2 — Comparison of movement of natural and synthetic mRNAs
To compare rate of movement of synthetic RNAs with natural mRNAs, oocytes were coinjected with Lys+ monomethyl cap RNA (5ng per oocyte)
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and P-reovlrus mRNA (60ng per oocyte) (fig. 30a). Because of the difference in the specific activity of the two types of RNA two
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P-labeiled Lys monomethyl cap RNA was injected (5ng/oocyte) into the animal (A inj) or vegetal pole (V inj) of oocytes. At 0 and 24 hours (h) after injection the oocytes were frozen and sectioned into their animal (A) and vegetal (V) halves. RNA extracted from the oocyte halves was run on a denaturing agarose/formaldehyde gel, which, after ethidium staining, was dried down and autoradiographed directly. Tracks (m) contain non-injected RNA (1, lng; 5, 5ng; 10, lOng).