Transfection of wild-type and 'Fanconi anemia-like' mouse lymphoma mutant cells by electroporation

An electroporation protocol for the successful transfection of mouse lymphoblastoid cells has been developed. Two cell lines, a normal and a mutant sensitive to DNA cross-linking agents, were used. The optimum conditions of electroporation in terms of uptake of the fluorescent dye lucifer yellow coupled with low toxicity were established. Subsequently, these conditions were used to achieve stable transfection by a plasmid expression vector. The plasmid integration patterns were determined by Southern blot analysis.     

Association of the type II cAMP-dependent protein kinase with a human thyroid RII-anchoring protein. Cloning and characterization of the RII-binding domain.

The type II cAMP-dependent protein kinase (PKA) is localized to specific subcellular environments through binding of the dimeric regulatory subunit (RII) to anchoring proteins. Subcellular localization is likely to influence which substrates are most accessible to the catalytic subunit upon activation. We have previously shown that the RII-binding domains of four anchoring proteins contain sequences which exhibit a high probability of amphipathic helix formation (Carr, D. W., Stofko-Hahn, R. E., Fraser, I. D. C., Bishop, S. M., Acott, T. E., Brennan, R. G., and Scott J. D. (1991) J. Biol. Chem. 266, 14188-14192). In the present study we describe the cloning of a cDNA which encodes a 1015-amino acid segment of Ht 31. A synthetic peptide (Asp-Leu-Ile-Glu-Glu-Ala-Ala-Ser-Arg-Ile-Val-Asp-Ala-Val-Ile-Glu-Gln-Val -Lys-Ala-Ala-Tyr) representing residues 493-515 encompasses the minimum region of Ht 31 required for RII binding and blocks anchoring protein interaction with RII as detected by band-shift analysis. Structural analysis by circular dichroism suggests that this peptide can adopt an alpha-helical conformation. Both Ht 31 (493-515) peptide and its parent protein bind RII alpha or the type II PKA holoenzyme with high affinity. Equilibrium dialysis was used to calculate dissociation constants of 4.0 and 3.8 nM for Ht 31 peptide interaction with RII alpha and the type II PKA, respectively. A survey of nine different bovine tissues was conducted to identify RII binding proteins. Several bands were detected in each tissues using a 32P-RII overlay method. Addition of 0.4 microM Ht 31 (493-515) peptide to the reaction mixture blocked all RII binding. These data suggest that all anchoring proteins bind RII alpha at the same site as the Ht 31 peptide. The nanomolar affinity constant and the different patterns of RII-anchoring proteins in each tissue suggest that the type II alpha PKA holoenzyme may be specifically targeted to different locations in each type of cell.     

Methylation patterns at the hypervariable X-chromosome locus DXS255 (M27 beta): correlation with X-inactivation status.

Methylation patterns surrounding a hypervariable X-chromosome locus, DXS255, have been analyzed with the restriction enzyme MspI and its methylation-sensitive isoschizomer HpaII. HpaII sites flanking the hypervariable region were found to be methylated on 41 active X chromosomes and unmethylated on 11 inactive X chromosomes present in a range of male, female, and hybrid cells and tissues. This differential methylation pattern coupled with the previously described high level (greater than 90%) of heterozygosity at the DXS255 locus can therefore be applied to determine the inactivation status of X chromosomes in females heterozygous for X-linked disease and in tumor clonality studies.