Disruption of the CAR1 Gene Encoding Arginase Enhances Freeze Tolerance of the Commercial Baker's Yeast Saccharomyces cerevisiae

ABSTRACT The effect of intracellular charged amino acids on freeze tolerance in doughs was determined by constructing homozygous diploid arginase-deficient mutants of commercial baker's yeast. An arginase mutant accumulated higher levels of arginine and/or glutamate and showed increased leavening ability during the frozen-dough baking process, suggesting that disruption of the CAR1 gene enhances freeze tolerance.

The CAR1 Gene Encoding a Cellular Receptor Specific for Subgroup B and D Avian Leukosis Viruses Maps to the Chickentvb Locus

ABSTRACT Host susceptibility to subgroup B, D, and E avian leukosis viruses (ALV) is determined by specific alleles of the chicken tvblocus. Recently, a chicken gene that encodes a cellular receptor, designated CAR1, specific for subgroups B and D ALV was cloned, and it was proposed that this gene was the s3 allele oftvb (J. Brojatsch, J. Naughton, M. M. Rolls, K. Zingler, and J. A. T. Young, Cell 87:845–855, 1996). We now report that in a backcross derived from an F1 (Jungle Fowl × White Leghorn [WL]) male mated with inbred WL females, the cloned ALV receptor gene cosegregated with two markers linked totvb. The two markers used were atvbs1 -specific antigen recognized by the chicken R2 alloantiserum and restriction fragment length polymorphisms associated with the expressed sequence tag com152e. With all three markers, no crossovers were observed among 52 backcross progeny tested and LOD linkage scores of 15.7 were obtained. These data demonstrate that CAR1 is the subgroup B and D ALV susceptibility gene located at tvbs3.

The cell density factor CMF regulates the chemoattractant receptor cAR1 in Dictyostelium.

Starving Dictyostelium cells aggregate by chemotaxis to cAMP when a secreted protein called conditioned medium factor (CMF) reaches a threshold concentration. Cells expressing CMF antisense mRNA fail to aggregate and do not transduce signals from the cAMP receptor. Signal transduction and aggregation are restored by adding recombinant CMF. We show here that two other cAMP-induced events, the formation of a slow dissociating form of the cAMP receptor and the loss of ligand binding, which is the first step of ligand-induced receptor sequestration, also require CMF. Vegetative cells have very few CMF and cAMP receptors, while starved cells possess approximately 40,000 receptors for CMF and cAMP. Transformants overexpressing the cAMP receptor gene cAR1 show a 10-fold increase of [3H]cAMP binding and a similar increase of [125I]CMF binding; disruption of the cAR1 gene abolishes both cAMP and CMF binding. In wild-type cells, downregulation of cAR1 with high levels of cAMP also downregulates CMF binding, and CMF similarly downregulates cAMP and CMF binding. This suggests that the cAMP binding and CMF binding are closely linked. Binding of approximately 200 molecules of CMF to starved cells affects the affinity of the majority of the cAR1 cAMP receptors within 2 min, indicating that an amplifying mechanism allows one activated CMF receptor to regulate many cARs. In cells lacking the G-protein beta subunit, cAMP induces a loss of cAMP binding, but not CMF binding, while CMF induces a reduction of CMF binding without affecting cAMP binding, suggesting that the linkage of the cell density-sensing CMF receptor and the chemoattractant cAMP receptor is through a G-protein.

Extracellular cAMP can restore development in Dictyostelium cells lacking one, but not two subtypes of early cAMP receptors (cARs). Evidence for involvement of cAR1 in aggregative gene expression

Extracellular cAMP induces expression of several classes of developmentally regulated genes in Dictyostelium. Four highly homologous surface cAMP receptors (cARs) were identified earlier, but involvement of specific cARs in gene regulation has not been clarified. Cells lacking the chemotactic receptor, cAR1, neither aggregate nor express developmentally regulated genes. Expression of aggregative genes is in wild-type cells induced by nanomolar cAMP pulses and repressed by persistent micromolar cAMP stimuli, which induce expression of prespore and prestalk-enriched genes during the postaggregative stages of development. We show here that in cell lines carrying a cAR1 gene disruption, nanomolar pulses cannot induce aggregative gene expression. Remarkably, micromolar cAMP can induce expression of aggregative genes in car1- cells as well as expression of prespore and prestalk-enriched genes, and furthermore restores their ability to form normal slugs and fruiting bodies. These data indicate that cAR1 mediates aggregative but not postaggregative gene expression and morphogenesis, and suggest that after gene disruption, its function is partially taken over by a lower affinity receptor that is not subjected to desensitization. The absence of another early cAMP receptor, cAR3, does not affect development. However, in a car1-/car3- double mutant, cAMP stimulation cannot restore any developmental gene expression, indicating that cAR3 may have substituted for cAR1 in car1- cell lines.