Multiple sex pheromones and receptors of a mushroom-producing fungus elicit mating in yeast.

The mushroom-producing fungus Schizophyllum commune has thousands of mating types defined, in part, by numerous lipopeptide pheromones and their G protein-linked receptors. Compatible combinations of pheromones and receptors encoded by different mating types regulate a pathway of sexual development leading to mushroom formation and meiosis. A complex set of pheromone-receptor interactions maximizes the likelihood of outbreeding; for example, a single pheromone can activate more than one receptor and a single receptor can be activated by more than one pheromone. The current study demonstrates that the sex pheromones and receptors of Schizophyllum, when expressed in Saccharomyces cerevisiae, can substitute for endogenous pheromone and receptor and induce the yeast pheromone response pathway through the yeast G protein. Secretion of active Schizophyllum pheromone requires some, but not all, of the biosynthetic machinery used by the yeast lipopeptide pheromone a-factor. The specificity of interaction among pheromone-receptor pairs in Schizophyllum was reproduced in yeast, thus providing a powerful system for exploring molecular aspects of pheromone-receptor interactions for a class of seven-transmembrane-domain receptors common to a wide range of organisms.

Scooter, a new active transposon in Schizophyllum commune, has disrupted two genes regulating signal transduction.

Two copies of scooter, a DNA-mediated transposon in the basidiomycetous fungus Schizophyllum commune, were characterized. Scooter is the first transposon isolated from S. commune. Scooter creates 8-bp target site duplications, comparable to members of the hAT superfamily, and has 32-bp terminal inverted repeats. Both copies of scooter are nonautonomous elements capable of movement. Southern blot hybridizations show that scooter-related sequences are present in all S. commune strains tested. Scooter-1 was identified initially as an insertion in the Bbeta2 pheromone receptor gene, bbr2, leading to a partial defect in mating. Scooter-2 spontaneously disrupted a gene to produce the frequently occurring morphological mutant phenotype known as thin. The scooter-2 insert permitted cloning of the disrupted gene, thn1, which encodes a putative regulator of G protein signaling (RGS) protein. Spontaneous insertion of scooter into genes with identifiable mutant phenotypes constitutes the first evidence of active transposition of a DNA-mediated transposon in a basidiomycete.

Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune.

Schizophyllum commune has thousands of mating types defined in part by numerous lipopeptide pheromones and their G-protein-coupled receptors. These molecules are encoded within multiple versions of two redundantly functioning B mating-type loci, B alpha and B beta. Compatible combinations of pheromones and receptors, produced by individuals of different B mating types, trigger a pathway of fertilization required for sexual development. Analysis of the B beta 2 mating-type locus revealed a large cluster of genes encoding a single pheromone receptor and eight different pheromones. Phenotypic effects of mutations within these genes indicated that small changes in both types of molecules could significantly alter their specificity of interaction. For example, a conservative amino acid substitution in a pheromone resulted in a gain of function toward one receptor and a loss of function with another. A two-amino-acid deletion from a receptor precluded the mutant pheromone from activating the mutant receptor, yet this receptor was activated by other pheromones. Sequence comparisons provided clues toward understanding how so many variants of these multigenic loci could have evolved through duplication and mutational divergence. A three-step model for the origin of new variants comparable to those found in nature is presented.

Tolosa-Hunt syndrome. The dangers of an eponym.

Two cases of painful ophthalmoplegia are described which were initially thought to be examples of the Tolosa-Hunt syndrome. Both were partially responsive to steroid treatment. Subsequent investigations showed that in one case the condition was due to an aneurysm and in the other to a malignant lymphoma.

Use of a personal computer to monitor adverse drug reactions.

The Pharmacy department at Sewickley Valley Hospital, Sewickley, Pennsylvania, has developed an adverse drug reaction database using DBase III Plus software. The computer database allows instant, custom manipulation of large amounts of data, and aids in the analysis of adverse drug reaction reports for quality assurance purposes.

Recovery of nerve conduction after a pneumatic tourniquet: observations on the hind-limb of the baboon.

A small pneumatic cuff inflated around the knee was used to produce tourniquet paralysis in baboons. A cuff pressure of 1,000 mm Hg maintained for one to three hours produced paralysis of distal muscles lasting up to three months. Nerve conduction studies showed that most of the motor fibres to the abductor hallucis muscle were blocked at the level of the cuff and that they conducted impulses normally in their distal parts. There was a significant correlation between the duration of compression and that of the subsequent conduction block. When tested two to three weeks after the tourniquet, the amplitude of the response of m. abductor hallucis to nerve stimulation distal to the cuff was usually slightly reduced compared with the precompression figure. This was assumed to mean that a small proportion of the motor fibres had undergone Wallerian degeneration as a result of compression. Maximal motor conduction velocity was reduced in recovering nerves. It was also reduced when a cuff pressure of 500 mm Hg was used, which was insufficient to produce persistent conduction block. In such cases a reduced velocity without evidence of block could be demonstrated 24 hours after compression. Ascending nerve action potentials were recorded from the sciatic nerve in the thigh, with stimulation at the ankle. Before compression the fastest afferent fibres had a significantly higher velocity than the fastest motor fibres in the same nerve trunk. Results after compression suggested that the high-velocity afferent fibres had a susceptibililty to the procedure similar to that of the fastest motor fibres.

Characterization and expression of four proline-rich cell wall protein genes in Arabidopsis encoding two distinct subsets of multiple domain proteins.

We have characterized the molecular organization and expression of four proline-rich protein genes from Arabidopsis (AtPRPs). These genes predict two classes of cell wall proteins based on DNA sequence identity, repetitive motifs, and domain organization. AtPRP1 and AtPRP3 encode proteins containing an N-terminal PRP-like domain followed by a C-terminal domain that is biased toward P, T, Y, and K. AtPRP2 and AtPRP4 represent a second, novel group of PRP genes that encode two-domain proteins containing a non-repetitive N-terminal domain followed by a PRP-like region rich in P, V, K, and C. Northern hybridization analysis indicated that AtPRP1 and AtPRP3 are exclusively expressed in roots, while transcripts encoding AtPRP2 and AtPRP4 were most abundant in aerial organs of the plant. Histochemical analyses of promoter/beta-glucuronidase fusions localized AtPRP3 expression to regions of the root containing root hairs. AtPRP2 and AtPRP4 expression was detected in expanding leaves, stems, flowers, and siliques. In addition, AtPRP4 expression was detected in stipules and during the early stages of lateral root formation. These studies support a model for involvement of PRPs in specifying cell-type-specific wall structures, and provide the basis for a genetic approach to dissect the function of PRPs during growth and development.

Deletion analysis and localization of SbPRP1, a soybean cell wall protein gene, in roots of transgenic tobacco and cowpea.

SbPRP1 is a member of the soybean (Glycine max L. Merr) proline-rich cell wall protein family and is expressed at high levels in root tissue. To characterize the sequences required for this expression, we have fused 1.1 kb of upstream flanking DNA sequence from an SbPRP1 genomic clone to a gene encoding beta-glucuronidase (GUS). This construct was introduced into tobacco using Agrobacterium tumefaciens-mediated transformation. Histochemical staining of GUS activity in transgenic tobacco indicated that SbPRP1 is expressed in the apical and elongating region of both primary and lateral roots, most strongly in the epidermis. A similar localization pattern was found in transformed hairy roots when this construct was introduced into cowpea (Vigna aconitifolia) using Agrobacterium rhizogenes-mediated transformation. Nested 5'-deletion analysis of the SbPRP1 promoter indicated that a minimal promoter for SbPRP1 expression in roots is located within the first 262 bases of upstream flanking DNA and that the region between -1080 and -262 is required for maximal expression of this gene. Gel retardation assays showed that nuclear factors can be detected in soybean roots which specifically bind to sequences located between -1080 and -623, a region which is needed for maximal expression of the SbPRP1 promoter. Northern hybridization analysis was also used to show that little SbPRP1 mRNA was present in roots during the first 24 h after imbibition. These studies indicate that SbPRP1 expression is localized to the actively growing region of the root and that this expression is temporally regulated during very early stages of seedling growth.

Expression of DcPRP1 is linked to carrot storage root formation and is induced by wounding and auxin treatment.

A carrot (Daucus carota, L.) genomic clone (DcPRP1) was isolated on the basis of its homology to previously described cDNAs encoding a wound-inducible, proline-rich cell wall protein. DNA sequence analysis showed that DcPRP1 contains a single open-reading frame encoding a 235-amino acid protein that is colinear with that predicted from the cDNA sequence with the exception of four amino acids at the N terminus and a 60-nucleotide insertion present within the genomic clone. Genomic Southern hybridization analysis showed that the cloned sequence hybridized with a single restriction enzyme fragment using several restriction enzymes. Primer extension and northern hybridization analysis indicated that the expression of DcPRP1 is developmentally regulated and linked to the formation of storage roots, where this gene is expressed at high levels after wounding. The level of DcPRP1 mRNA was greatest in tissue immediately adjacent to the wound site. Treatment of unwounded carrot storage roots with 10 microM 2,4-dichlorophenoxy-acetic acid, indoleacetic acid, or naphthalene-1-acetic acid also resulted in the accumulation of DcPRP1 transcripts to a level equal to that seen in wounded tissue.