Xenopus system isolation

Other phosphatases have also been identified and may be implicated in mitotic and meiotic germ cell functions. For example, INH was originally isolated from a Xenopus oocyte cell free system as an inhibitor of pre-MPF activity (Cyert and Kirschner, 1988). INH encodes a protein phosphatase 2Athat negatively regulates MPF activity by dephosphory-lating Cdc2 on thr-161 (Lee et al., 1991 Solomon et al., 1990). 

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. 

The Xenopus oocyte expression system can be used for characterization of products of specific mRNAs transcribed in vitro from cDNA isolates. Expression cloning of novel cDNAs, whose function can be assayed following expression, can also be performed using oocytes. In the latter case, as shown in Eig. 4.3, total mRNA is fractionated by size, and expression in oocytes is used to identify mRNA fractions capable of producing the protein of interest. The enriched mRNA fractions are used for cDNA preparation. The cDNA of interest is identified by its ability to select (by hybridization) its mRNA from the total mRNA pool. The selected mRNA is assayed again in oocytes, as shown in Fig. 4.3. Repeated rounds of this procedure with further enrichment of the desired cDNA from the pool leads to the isolation of the cDNA. Alternatively, in vitro transcription of the cDNA pool can also be used to generate mRNA for injection and assay. Component cDNAs of the pool that... 

Recombinant DNA methods have been extensively used to isolate and analyze the sequence of numerous receptors from various mammalian complimentary DNA (cDNA) libraries using the polymerase chain reaction (PCR) technique (98) to clone receptors that can then be expressed in various pro- and eukaryotic cell lines and Xenopus oocyte. Conserved regions in receptors that are involved in ligand binding, coupling to transductional systems and ion channel formation, have thus been identi-... 

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