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The type RIIβ regulatory subunit of protein kinase A is primarily expressed in adipose tissue and brain. Knockout mice suggest a role for RIIβ in regulating energy balance and adipose-tissue content, thus making it a potential target for therapeutic intervention in obesity. A truncated version of the RIα subunit has been used in a crystallographic study and was used here to design an analogous RIIβ construct. Despite substantial screening, conditions were not found for the crystallization of the truncated RIIβ subunit. However, limited proteolysis of the full-length RIIβ subunit identified boundaries of the 'hinge' region and a fragment containing the two cAMP-binding domains which did crystallize. A recombinant version of the fragment was expressed and crystallized for X-ray diffraction studies. The crystals belong to the orthorhombic space group C222, with unit-cell parameters a = 91.6, b = 105.9, c = 85.8 Å , and diffracted to at least 2.3 Å. [ABSTRACT FROM AUTHOR]

Diphtheria toxin-resistance markers in two translational mutants, CH-RE1.22c, possessing no toxin-sensitive EF-2 (class IIa), and CH-RE1.32, with 50% toxin-sensitive and 50% toxin-resistant EF-2 (class IIb), behaved codominantly in somatic cell hybrids. There was no complementation in hybrids formed between the two resistant mutants. The mutant parents and their hybrids, except those formed by fusion of CH-RE1.32 and wild-type cells, grew in the presence of toxin. To explain these results we suggest that CHO-K1 cells possess two functional copies of the gene for EF-2 and that CH-RE1.22c and CH-RE1.32 represent the homozygous (R/R) and heterozygous (R/S) states of resistance at the EF-2 gene locus. The failure of hybrids formed between CH-RE1.32 and wild-type cells to grow in toxin is a gene dosage effect. Codominant class IIa translational resistance is a selectable marker for the isolation of hybrids. It can be combined with a second, recessive, marker to provide a cell which is a 'universal hybridizer' (10). [ABSTRACT FROM AUTHOR]