Xenopus isolation

Allen, D.G., Lee, J.A., Westerblad, H, (1989). Intracellular calcium and tension in isolated single muscle fibers from Xenopus. J. Physiol. 415,433-458. 

Zasloff, M. Magainins, a class of antimicrobial peptides from Xenopus skin Isolation, characterization of two active forms, and partial cDNA sequence of a precursor. Proc. Nad. Acad. Sci. USA 1987, 84, 5449-5453. 

Expression studies in Xenopus oocytes or transfected cell lines originally suggested that functional GABA-activated chloride channels could be formed by receptor subunits of each class in isolation. However, much better expression occurs with two or more subunit types in combination and it is likely that most native receptors contain at least three different subunits. Co-expression of a and /I subunits results in the assembly of... 

Other recent and very compelling evidence that the P subunit may also be necessary for full activity [83] comes from the study of a high threshold but DHP-insen-sitive brain channel that has an a 1-like channel core. The cDNA for this brain channel was recently isolated and the mRNA was expressed in Xenopus oocytes. The current corresponding to this channel was increased when the mRNA for either the skeletal muscle 0.2 or P subunits were co-injected. However, when the brain ai-like mRNA was co-expressed with the combination of the skeletal muscle 2 and p mRNAs, the current was dramatically increased from 31 nA for the brain ai alone to 6 500nA for the combination [83]. These striking results are the best evidence so far obtained that the P subunit has a functional role in channel activity. Although these data were obtained with a DHP-insensitive channel, they pro-... 

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). 

Control of the onset of transcription. Cell 30 687—696 Robertson K, Hensey C, Gautier J 1999 Isolation and characterization of Xenopus ATM (X-ATM) expression, localization, and complex formation during oogenesis and early development. Oncogene 18 7070-7079... 

Boumah, C. E., et al. Functional expression of the nitrobenzylthioino-sine-sensitive nucleoside transporter of human choriocarcinoma (BeWo) cells in isolated oocytes of Xenopus laevis. Biochem. J. 1994, 299, 769-773. 

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. 

In vitro transcription from cloned cDNA sequences is required to produce mRNA that can be injected into Xenopus oocytes for expression of the encoded protein and is achieved by standard procedures. While oocyte isolation and injection generally follows the protocols provided below, in vitro transcription into mRNA may be modified in accordance to the instructions provided with... 

The ovaries of a fully mature Xenopus frog contain upward of 10,000 oocytes. Hundreds of viable oocytes can be isolated from a donor frog and the same frog can be reused. 

Numerous protocols describing the care and maintenance of Xenopus laevis frogs and the isolation of oocytes from their ovaries have been published (Cohnan, 1984 Goldin, 1992 Quick and Lester, 1994 Stiihmer and Parekh, 1995 Theodoulou and Miller, 1995). The following methods are based primarily on the procedures described by Yao and colleagues (2000). 

Using optical traps, Cui and Bustamante [76] stretched isolated chicken erythrocyte fibers, and Bennink et al. [77] pulled on fibers directly reconstituted in the flow cell from X-DNA and purified histones with the help of Xenopus extracts (see Fig. 10a for a schematic of the latter experiment). Up to 20 pN, the fibers underwent reversible stretching, but applying stretching forces above 20 pN led to irreversible alterations, interpreted in terms of removal of histone octamers from the fibers with recovery of the mechanical properties of naked DNA. 

Although absolute proof is still lacking it seems clear that NTCP is the major sodium-dependent transporter of bile acids, although a minor role for other proteins cannot be excluded. It has now been isolated from rat, mouse, rabbit and human. The rat polypeptide was first expressed in Xenopus laevis oocytes and shown to be a 362 amino acid glycoprotein with 7 or 9... 

The requirement for zinc in the regulation of gene expression is exemplified by transcription factor IIIA, which has been shown to contain from two (Hanas et al., 1983) to seven to eleven (Miller et al., 1985) zinc ions bound to a 40K protein molecule. Transcription factor IIIA is obtained from immature Xenopus oocytes in a complex with 5 S RNA, and the protein is required for transcription initiation the apoprotein does not bind to the 5 S RNA gene (Hanas et al, 1983). Uncertainties regarding the stoichiometric requirement for zinc persist, given the report by Shang et al. (1989) that transcription factor IIIA, as either the isolated protein or the 5 S RNA complex, contains two firmly bound zinc ions which are required for transcription activation. 

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