Embryo immunoblotting

There is a report that in pea embryo, some of the SSSs may also be bound to the starch granule (Denyer et al., 1993) and that in maize endosperm, some of SSSI adheres to the starch granule (Mu et al., 1994). The conclusions in the pea embryo study (Denyer et al., 1993) are based on positive immunoblots obtained after electrophoresis of the SSS with antibody prepared against the GBSS, and also on the similarity of the amino-acid sequence of three peptides obtained from protease SV8 digests of the SSS. This clearly shows there is a close relationship between SSSII and GBSSII, but does not indicate that they are identical proteins. It is also... 

Fig. 4 Immunoblot analysis demonstrating mobility differences among different Drosophila lamin isoforms. Lane 1, the 20-cm long 7% polyacrylamide gel was loaded with stage 14 oocyte extract containing only lamin Dm ,i, lane 2, loaded with embryo nuclei containing both lamins Dmi and Dm2. The migration positions of the different lamin isoforms are indicated to the right of the figure.

Additional biochemical characterization of in vitro disassembly products can be achieved through two-dimensional gel analysis. After either silver staining (Maus et ai, 1995) or immunoblot analysis (Smith et al., 1987 Havel et at., 1992), interphase lamins Dmi and Dm2 can be detected as multiple spots, indicating multiple posttranslationally modified forms. Meiotic lamin Dmmit in the stage 14 oocyte extract exhibits an entirely different pattern (Maus et ai, 1995). When embryo nuclei, for example, are added to an oocyte disassembly extract and incubated for 90 min, a pattern distinct from the interphase lamins and indistinguishable from that of the extract alone should be seen, indicating that embryo nuclear lamin was converted to forms highly similar to, if not identical to, meiotic lamin Dm ,ii. 

Some degree of controversy exists as to whether nuclear envelope formation precedes, parallels, or follows the assembly of a nuclear lamina (see Georgatos et ai, 1994 Lourim and Krohne, 1994). Formation of the nuclear envelope in Drosophila embryo extracts is lamin dependent (Ulitzur et ai, 1992). In contrast, lamin-independent nuclear envelope assembly in vitro has been reported in Xenopus (Newport et ai, 1990 Meier et ai, 1991) and sea urchin (Collas et ai, 1995). The latter studies corroborate immunofluorescence observations of nuclear reconstitution after mitosis in somatic mammalian cells in vivo (Chaudhary and Courvalin, 1993). Assembly of a nuclear lamina in vitro can be monitored by immunofluorescence and immunoblotting using anti-lamin antibodies. 

We had shown previously that the phorbolester promoter TPA increased poly(ADP-ribosyl)ation of e same histones in mouse embryo fibroblasts C3H10T1/2 but in this case H2B was the major acceptor (38). H2B was also the major poly(ADP-ribose) acceptor in Yoshida hepatoma cells following exposure to the methylating agent dimethylsulfate (32). However, it should be recognized that no immunoblots were used for the identification of the individual histones in the previous woik. 

Immunoblotting of Proteins from Single Drosophila Embryos ... 

The first method takes advantage of the cortical location of mitoses 11-13. Edgar et al. (1994) first identified live embryos that were undergoing mitosis by the disappearance of the nuclear envelope under DIG (differential interference contrast) optics. The embryos were then aged for appropriate time intervals to reach desired points in the following division cycle, prior to homogenization. Half of the extract from one embryo was used for HI kinase assays and the other half was immunoblotted to correlate kinase activity with phosphorylation on Cdkl and with cyclin levels. 

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