Antibody oocytes

Mailer Our original model was based on data that J. Tata published showing that bcl2 was one of the first genes transcribed by the embryo. This was a sort of default model where unless you produced zygotic Bcl2 you would apoptose. But when we made antibodies to Bcl2 we found that there was plenty of it present in the oocyte. 

Kubiak I don t know what happens to the centriole material that is brought by the sperm. What we detect at the spindle pole is a material that is reactive to y-tubulin or MPM2 antibodies. We call it PCM because the same material accompanies centrioles in other cells. All that we know is that there are plenty of these small spots in mouse oocytes also out of the spindle and that they can be gathered to opposite places with respect to the chromosomes. Moreover, as I mentioned, we have shown recently that this also happens in the absence of chromosomes (Brunet et al 1998). 

Because the fMet-Leu-Phe receptor is present only at low levels in neutrophils (-12 x 10 15 g of receptor per cell), it has proved difficult to purify and characterise. Researchers have therefore turned to molecular cloning techniques to gain insight into the molecular structure of this receptor. This approach itself has not been easy because, in the absence of an antibody that specifically binds to the receptor, or else without some amino acid sequence data that can be used to synthesise oligonucleotide probes, cDNA libraries cannot be screened to isolate relevant clones. Therefore, experimental systems in which functional fMet-Leu-Phe receptors are expressed on the surfaces of transfected cells have been used. Two main systems have been utilised expression of mRNA injected into Xenopus laevis oocytes and cDNA cloning into the COS-cell expression vector. 

Incubated oocytes accept hydrocarbons at similar rates from hemolymph and from purified HDLp, suggest that LTP might not be required for uptake of hydrocarbons (Fan et al., 2002). However, this idea will have to await careful experiments with LTP antibodies, as detailed above. In M. sexta LTP plays a role in delivery of lipids to the developing oocytes and the conversion of adult HDLp to VHDLp in the egg (Liu and Ryan, 1991). 

Portion of a "lampbrush chromosome" from an oocyte of the newt Nophthalmus viridescens] hnRNP protein associated with nascent RNA transcripts fluoresces red after staining with a monoclonal antibody. [Courtesy... 

Currently, it is standard procedure to develop ion channel-specific antibodies for immunocytochemistry, to perform Western and Northern blot analyses, ion channel in situ hybridization, or reverse transcription polymerase chain reaction (RT-PCR). The introduction of the single-cell RT-PCR in combination with the patch-clamp method in the 1990s made it possible to identify gene transcripts and to correlate them with functional data for the same individual cell. Finally, one of the most powerful cell biological techniques in the study of ion channels is based on artificial expression systems such as microinjection of mRNA encoding channel subunits into Xenopus oocytes and selective expression of native ion channels or with different subunit composition (e.g., Ky channel subunits). Because the Xenopus oocytes are large, they are a perfect model to study artificially expressed channels. Another good model for artificial ion channel expression is the Chinese hamster ovary (CHO) cell line. 

Various proteins involved in cell—cell (e.g., sperm-oocyte), virus-cell, bactmum-ceH, and hormone—cell interactions C tain plasma proteins of coldwat fish Lectins, selectins (cell adhesion lectins), antibodies Various proteins involved in hwmone and drug action Calnexin, calreticulin... 

Microinject a freshly isolated oocyte with 100-200 nl of an antinucleoporin antibody conjugated with 8-nm colloidal gold. 

Immunoprecipitation analysis can be used to determine if a specific protein, for which an antibody is available, becomes associated with a microinjected RNA. Moreover, antibodies against particular RNA modifications (e.g., 5 cap structures or modified bases) can be employed to determine if an injected RNA undergoes a particular posttranscriptional modification following oocyte injection. 

Preparation of oocyte samples and immunoprecipitation. Extracts containing P-labeled RNAs are prepared either from whole oocytes or isolated nuclear and cytoplasmic fractions by homogenization on ice in a suitable buffer (e.g., 40 mM HEPES, pH 7.9, with KOH 100 mM potassium acetate, 8% (v/v) glycerol, 5 mM MgS04, 2 mM EGTA, 0.2 mM EDTA, 1 mM dithiothreitol, 0.5% (v/v) aprotinin, 2 /ig/ml of each leupeptin and pepstatin, and 0.1 U/jul of RNasin) at a ratio of at least 25 pi of buffer per oocyte (or isolated cytoplasm) or as little as 1-4 pi per isolated nucleus. If the antibody recognizes RNA rather than an... 

Generally, one oocyte equivalent of extract (or extracted RNA) is added to the antibody-coupled beads and brought to 0.25 ml with Ippiso (the same composition as Ippsoo except having 150 mM NaCl) with 0.1 U/ml RNasin and 2 mM dithiothreitol. Ippsoo can be used in this and all subsequent steps for salt-stable RNA/protein interactions and will increase the stringency of precipitation. Precipitations should be carried out for between 2 hr and overnight at 4°C with end-over-end rotation. 

An example of an RNA injection/immunoprecipitation experiment is shown in Fig. 2. In this experiment, a mixture of radiolabeled snRNAs were coinjected into oocyte nuclei. Oocyte fractionation over time followed by electrophoresis of the RNAs in both the nuclear and cytoplasmic compartments (Fig. 2A) revealed the nucleocytoplasmic distributions of the injected RNAs. The RNAs present in the nuclear fractions were extracted and immunoprecipitated (Fig. 2B and C) using antibodies recognizing either the m G cap (of the precursor snRNAs) or a hypermethylated m G cap (of the mature snRNAs). This and other experiments (Terns and Dahlberg, 1994 Terns et ai, 1995) demonstrated that U3 RNA and other small nucleolar RNAs differ from most RNA polymerase Il-transcribed RNAs in that they are not exported from the nucleus to the cytoplasm. Furthermore, unlike spliceosomal RNAs, which undergo hypermethy-lation of their m G cap structures within the cytoplasm (Mattaj, 1986), small nucleolar RNAs receive their trimethylated cap structures within the cell nucleus. 

Erikson E, Stefanovic D, Blenis J, Erikson RL, Mailer JL. 1987. Antibodies to Xenopus egg S6 kinase II recognize S6 kinase from progesterone- and insulin-stimulated Xenopus oocytes and from proliferating chicken embryo fibroblasts. Mol Cell Biol 7(9) 3147-3155. 

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