Ubiquitylation of the transducin betagamma subunit complex. Regulation by phosducin

G proteins (Galphabetagamma) are essential signaling molecules, which dissociate into Galpha and Gbetagamma upon activation by heptahelical membrane receptors. We have identified the betagamma subunit complex of the photoreceptor-specific G protein, transducin (T), as a target of the ubiquitin-proteasome pathway. Ubiquitylated species of the transducin gamma-subunit (Tgamma) but not the alpha- or beta-subunits were assembled de novo in bovine photoreceptor preparations. In addition, Tgamma was exclusively ubiquitylated when Tbetagamma was dissociated from Talpha. Ubiquitylation of Tbetagamma on Tgamma was selectively catalyzed by human ubiquitin-conjugating enzymes UbcH5 and UbcH7 and was coincident with degradation of the entire Tbetagamma subunit complex in vitro by a mechanism requiring ATP and the proteasome. We also show that Tbetagamma association with phosducin, a photoreceptor-specific protein of unknown physiological function, blocks Tbetagamma ubiquitylation and subsequent degradation. Phosphorylation of phosducin by Ca(2+)/calmodulin-dependent protein kinase II, which inhibits phosducin-Tbetagamma complex formation, completely restored Tbetagamma ubiquitylation and degradation. We conclude that Tbetagamma is a substrate of the ubiquitin-proteasome pathway and suggest that phosducin serves to protect Tbetagamma following the light-dependent dissociation of Talphabetagamma.     

Lysophospholipid Receptors in the Nervous System

The lysophospholipid mediators, lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), are responsible for cell signaling in diverse pathways including survival, proliferation, motility, and differentiation. Most of this signaling occurs through an eight-member family of G-protein coupled receptors once known as the endothelial differentiation gene (EDG) family. More recently, the EDG receptors have been divided into two subfamilies: the lysophosphatidic acid subfamily, which includes LPA₁, (EDG-2/VZG-1), LPA₂ (EDG-4), and LPA₃ (EDG-7), and the sphingosine-1-phosphate receptor subfamily, which includes S1P₁ (EDG-1), S1P₂ (EDG-5/H218/AGR16), S1P₃ (EDG-3), S1P₄ (EDG-6), and S1P₅ (EDG-8/NRG-1). The ubiquitous expression of these receptors across species, coupled with their diverse cellular functions, has made lysophospholipid receptors an important focus of signal transduction research. Neuroscientists have recently begun to explore the role of lysophospholipid receptors in a number of cell types; this research has implicated these receptors in the survival, migration, and differentiation of cells in the mammalian nervous system.     

Structure-function defects of human mitochondrial DNA polymerase in autosomal dominant progressive external ophthalmoplegia

Progressive external ophthalmoplegia (PEO) is a mitochondrial disorder associated with mutations in the POLG gene encoding the mitochondrial DNA polymerase (pol gamma). Four autosomal dominant mutations that cause PEO encode the amino acid substitutions G923D, R943H, Y955C and A957S in the polymerase domain of pol gamma. A homology model of the pol gamma catalytic domain in complex with DNA was developed to investigate the effects of these mutations. Two mutations causing the most severe disease phenotype, Y955C and R943H, change residues that directly interact with the incoming dNTP. Polymerase mutants exhibit 0.03-30% wild-type polymerase activity and a 2- to 35-fold decrease in nucleotide selectivity in vitro. The reduced selectivity and catalytic efficiency of the autosomal dominant PEO mutants predict in vivo dysfunction, and the extent of biochemical defects correlates with the clinical severity of the disease.     

Identification of domain boundaries within the N-termini of TAP1 and TAP2 and their importance in tapasin binding and tapasin-mediated increase in peptide loading of MHC class I

Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.     

Examination of the roles of Sgs1 and Srs2 helicases in the enforcement of recombination fidelity in Saccharomyces cerevisiae

Mutation in SGS1, which encodes the yeast homolog of the human Bloom helicase, or in mismatch repair (MMR) genes confers defects in the suppression of mitotic recombination between similar but nonidentical (homeologous) sequences. Mutational analysis of SGS1 suggests that the helicase activity is required for the suppression of both homologous and homeologous recombination and that the C-terminal 200 amino acids may be required specifically for the suppression of homeologous recombination. To clarify the mechanism by which the Sgs1 helicase enforces the fidelity of recombination, we examined the phenotypes associated with SGS1 deletion in MMR-defective and recombination-defective backgrounds. Deletion of SGS1 caused no additional loss of recombination fidelity above that associated with MMR defects, indicating that the suppression of homeologous recombination by Sgs1 may be dependent on MMR. However, the phenotype of the sgs1 rad51 mutant suggests a MMR-independent role of Sgs1 in the suppression of RAD51-independent recombination. While homologous recombination levels increase in sgs1Delta and in srs2Delta strains, the suppression of homeologous recombination was not relaxed in the srs2 mutant. Thus, although both Sgs1 and Srs2 limit the overall level of mitotic recombination, there are distinct differences in the roles of these helicases with respect to enforcement of recombination fidelity.     

Autosomal recessive inheritance of congenital goiter in Afrikander cattle

Congenital goiter was reported in a number of herds of Afrikander cattle in the 1950's. Some affected animals were assembled and maintained as a closed herd. Although considerable biochemical research into the nature of the disease has been conducted, no definitive report has described the mode of inheritance of the defect. This paper presents the results of breeding studies that indicate the defect is inherited as an autosomal recessive. Southern blot analysis of the thyroglobulin gene confirms this finding. In addition, serum levels of TSH (thyroid stimulating hormone, thyrotropin), T3 (3,4,3'-tri-iodothyronine), T4 (thyroxine), rT3 (3,3',5'-tri-iodothyronine), and DIT (diiodotyrosine) of goitrous animals are compared with normal animals.     

Two new polymorphic markers in the human proα2(1) collagen gene

Summary Structural defects in the human type 1 collagen genes are known to be the cause of several inherited disorders of connective tissue, such as osteogenesis imperfecta. The analysis and prenatal diagnosis of these disorders would be facilitated by establishing a set of polymorphic markers at these gene loci. We have previously reported the presence of an Msp 1 restriction fragment length polymorphism in the pro2(1) collagen genes of several Southern African populations (Grobler-Rabie et al., in press). This report describes the detection of a Bgl II and an EcoRI polymorphism in the pro2 gene of South African Blacks.     

Defective splicing of thyroglobulin gene transcripts in the congenital goitre of the Afrikander cattle

The structure of thyroglobulin mRNA was analyzed in an inbred herd of Afrikander cattle with hereditary goitre. Northern transfer of RNA from affected animals revealed both a shorter (approximately 7100 bases) and a normal-sized (approximately 8200 bases) thyroglobulin mRNA when hybridized to bovine thyroglobulin cDNA clones. S1 nuclease mapping experiments established that 1100 bases are deleted in the 5' region of the smaller mRNA. Electron microscopy of RNA from animals with goitre hybridized to a bovine genomic DNA clone showed that the region deleted corresponds to exon 9 of the thyroglobulin gene. Southern blot analysis of the exon 9 region revealed differences between affected and control animals with the enzymes PstI and TaqI. Although they could reflect a linkage disequilibrium between the mutation and restriction fragment length polymorphism, it is noteworthy that these differences map in the region of the exon 9/intron 9 junction. Our results show that a genetic lesion in the thyroglobulin gene causes aberrant splicing of the pre-mRNA, and suggest that the responsible mutation is at the exon 9/intron 9 junction.     

Thermal responses and survival after heat exposure are modulated by maintained differences in body temperature in mice

When individual mice were examined, it was found that the colonic body temperatureT _col of each individual within a genetically heterogeneous population tended to remain either above (warm) or below (cool) the population mean.T _col of warm, but not cool, mice showed circadian variation. When exposed to aT _a of 43° C, theT _col of cool mice increased by as musch as 2.4° C more than that of warm mice for a given 15 min increment of heating at 43°C. Survival of mice after acute lethal heat load (LD₇₅, –45°C) was significantly inversely correlated withT _col. Small persistent differences in body temperature of individuals may indicate differing thermal adaptedness.     

Marking and monitoring studies of the Kerguelen stock of southern elephant seals Mirounga leonina and their bearing on biological research in the Vestfold Hills

Southern elephant seals Mirounga leonina breed and moult on many subantarctic islands during the austral spring and summer. In the Kerguelen Province the subpopulations of M. leonina at Kerguelen (49°21S, 70°12E), Marion (46°54S, 37°45E) and Possession (46°25S, 51°45E) Islands have declined since 1970 and their present status at Heard Island (53°05S, 73°30E) is unknown. Population studies during their terrestrial phase have failed to explain the declines. Long distance movements of individuals between the subpopulations in question and also the Vestfold Hills, Antarctica (68°35S, 77°58E) have been recorded. The availability of food resources, competition with rapidly increasing fur seal populations and competition with fishing fleets have all been implicated in their decline. These explanations assume that communal feeding grounds are utilized. As they are predators entirely dependent on marine feeding, a study of their spatial and temporal distribution during their pelagic existence is of the utmost importance. Parameters describing growth, reproduction rates, population dynamics, and feeding ecology of the subpopulations in the Kerguelen Province may furthermore serve as indices of change within the marine ecosystem. The presence of a relatively large and predominantly male nonbreeding population of M. leonina at the Vestfold Hills in Antarctica which originates from the Kerguelen/Heard Island group, and which shows annual return, should be included in the marking and monitoring studies of the Kerguelen stock of southern elephant seals. Studies here, including an update of the size and social structure of the Heard island subpopulation, may elucidate the observed decline of, in particular, the adult bull component of the breeding stock.