Differential expression of retinoic acid receptor-beta isoforms during chick limb ontogeny.

Retinoids influence both morphogenetic events and differentiation during development of the vertebrate limb. These effects are mediated through nuclear retinoid receptors, which modulate target gene expression. We report here the cloning and characterization of three promoter- and splicing-variants of the retinoic acid receptor-beta (RAR-beta) from chick. These receptor isoforms are independently expressed during limb development. RAR beta 2 but not RAR beta 1 transcripts are enriched three-fold in the posterior limb bud, reflecting the increased RA concentrations in this region. RAR beta 1 transcripts are initially present throughout the limb bud mesenchyme and ectoderm, then become restricted within perichondrial regions and loose connective tissue of the limb. RAR beta 1 expression closely overlaps that of NCAM (neural cell adhesion molecule) and tenascin in non-neuronal tissues. RAR beta 2 transcripts are present within a subset of those limb tissues which express RAR beta 1. In the early limb bud RAR beta 2 transcripts are detected in proximal limb mesenchyme and in the initial mesenchymal condensate. In older limbs RAR beta 2 mRNAs are abundant in cells lateral to the digit cartilage. Neither RAR beta 1 nor RAR beta 2 transcripts are associated specifically with regions of limb cell death. The differential expression and regulation of RAR beta isoforms suggests these variants may have different roles in limb development.

Connections between the Ras-cyclic AMP pathway and G1 cyclin expression in the budding yeast Saccharomyces cerevisiae.

We have identified two processes in the G1 phase of the Saccharomyces cerevisiae cell cycle that are required before nutritionally arrested cells are able to return to proliferative growth. The first process requires protein synthesis and is associated with increased expression of the G1 cyclin gene CLN3. This process requires nutrients but is independent of Ras and cyclic AMP (cAMP). The second process requires cAMP. This second process is rapid, is independent of protein synthesis, and produces a rapid induction of START-specific transcripts, including CLN1 and CLN2. The ability of a nutritionally arrested cell to respond to cAMP is dependent on completion of the first process, and this is delayed in cells carrying a CLN3 deletion. Mating pheromone blocks the cAMP response but does not alter the process upstream of Ras-cAMP. These results suggest a model linking the Ras-cAMP pathway with regulation of G1 cyclin expression.

Changes in gene expression in the Ras/adenylate cyclase system of Saccharomyces cerevisiae: correlation with cAMP levels and growth arrest.

Levels of cyclic 3',5'-cyclic monophosphate (cAMP) play an important role in the decision to enter the mitotic cycle in the yeast, Saccharomyces cerevisiae. In addition to growth arrest at stationary phase, S. cerevisiae transiently arrest growth as they shift from fermentative to oxidative metabolism (the diauxic shift). Experiments examining the role of cAMP in growth arrest at the diauxic shift show the following: 1) yeast lower cAMP levels as they exhaust their glucose supply and shift to oxidative metabolism of ethanol, 2) a reduction in cAMP is essential for traversing the diauxic shift, 3) the decrease in adenylate cyclase activity is associated with a decrease in the expression of CYR1 and CDC25, two positive regulators of cAMP levels and an increase in the expression of IRA1 and IRA2, two negative regulators of intracellular cAMP, 4) mutants carrying disruptions in IRA1 and IRA2 were unable to arrest cell division at the diauxic shift and were unable to progress into the oxidative phase of growth. These results indicate that changes cAMP levels are important in regulation of growth arrest at the diauxic shift and that changes in gene expression plays a role in the regulation of the Ras/adenylate cyclase system.

Interactions between adenylate cyclase and the yeast GTPase-activating protein IRA1.

The adenylate cyclase system of the yeast Saccharomyces cerevisiae contains many proteins, including the CYR1 polypeptide, which is responsible for catalyzing the formation of cyclic AMP from ATP, RAS1 and RAS2 polypeptides, which mediate stimulation of cyclic AMP synthesis by guanine nucleotides, and the yeast GTPase-activating protein analog IRA1. We have previously reported that adenylate cyclase is only peripherally bound to the yeast membrane. We have concluded that IRA1 is a strong candidate for a protein involved in anchoring adenylate cyclase to the membrane. We base this conclusion on the following criteria: (i) a disruption of the IRA1 gene produced a mutant with very low membrane-associated levels of adenylate cyclase activity, (ii) membranes made from these mutants were incapable of binding adenylate cyclase in vitro, (iii) IRA1 antibodies inhibit binding of adenylate cyclase to the membrane, and (iv) IRA1 and adenylate cyclase comigrate on Sepharose 4B.

Agglutination of Erwinia stewartii Strains with a Corn Agglutinin: Correlation with Extracellular Polysaccharide Production and Pathogenicity.

A bacterial agglutinin was extracted from ground corn (WI hybrid 64A x W117) seed with phosphate-buffered saline (pH 6.0) and precipitated with (NH(4))(2)SO(4) at 70% saturation. The activities of this agglutinin against 22 strains of Erwinia stewartii (agent of bacterial wilt of corn) that varied in virulence were determined. Specific agglutination (agglutination titer per milligram of protein per milliliter) values were correlated negatively with virulence ratings. Strains with high specific agglutination values (15 or higher) were avirulent or weakly virulent; strains with low specific agglutination values (10 or lower) were highly virulent, with two exceptions. Avirulent strains produced butyrous colonies and released only small amounts of extracellular polysaccharide (EPS) into the medium, and the cells lacked capsules; virulent strains produced fluidal colonies and released large amounts of EPS, and the cells were capsulated. There was a strong correlation between the amount of EPS produced by each strain (as determined by increase in viscosity of the medium) and the specific agglutination value; in contrast, lipopolysaccharide compositions were similar in all strains. When cells of six fluidal strains were washed by repeatedly centrifuging and resuspending them in buffer, they were agglutinated more strongly by corn agglutinin than were unwashed cells. When avirulent cells were washed, their specific agglutination values did not increase significantly. Eight EPS-deficient mutants of E. stewartii, selected for resistance to the capsule-dependent bacteriophage K9, had lower virulence but higher specific agglutination than did their corresponding wild-type parents. Production of EPS appears to be essential for virulence; EPS may prevent agglutination of bacteria in the host, thus allowing their multiplication.