Tobacco Transformants Bearing Antisense Suppressor of Proline Dehydrogenase Gene, Are Characterized by Higher Proline Content and Cytoplasm Osmotic Pressure

The antisense suppressor was constructed for proline dehydrogenase gene (PDH; a fragment of PDH from Arabidopsis in antisense orientation and under the control of 35S promoter of cauliflower mosaic virus, CMV). In Nicotiana tabacum SR1 tobacco transformants bearing antisense suppressor for PDH, the proline content and the cytoplasm osmotic pressure were increased. The proline content in these transformants varied, whereas cytoplasm osmotic pressure was stable, which seems to reflect complicated relationships between these characteristics of the plant cell.     

Revertants and Secondary arom-2 Mutants Induced in Non-Complementing Mutants in the arom Gene Cluster of Neurospora Crassa

Extensive genetical and biochemical studies have been performed with revertants and secondary arom-2 mutants induced in two different primary non-complementing mutants which map within the arom gene cluster of Neurospora crassa. These studies indicate that mutant M54 but not M25 can revert by super-suppressor mutations in unlinked genes, thus confirming previous evidence that M54 contains a nonsense codon. At least three new super suppressors of M54 have been detected. All four super suppressors (including one previously detected) when combined with M54 result in high levels of all five of the arom enzymic activities in the form of arom multienzyme complexes very similar to (but not necessarily identical with) that in wild type (WT).-Evidence has also been obtained that the two non-complementing mutants can yield revertants which appear to result from true back mutations and produce arom aggregates essentially indistinguishable from that of WT. In addition, M25, but not M54, when plated on quinic acid yields revertants (secondary mutants) some of which are phenotypically indistinguishable from arom-2 primary mutants and others of which, although also mapping within the arom-2 gene, exhibit unusual properties. Genetic evidence indicates that the M25 secondary mutants are localized within the arom-2 gene, but that they arise from mutational events more complex than ones resulting in single base pair changes in the M25 codon.-The recovery of secondary arom-2 mutants as revertants of non-complementing arom mutants provides strong evidence, independent of earlier recombination data, that non-complementing arom mutants are located within the arom-2 structural gene of the arom gene cluster. In addition, the occurrence and characteristics of these secondary arom-2 mutants provide strong evidence, independent of the results with nonsense suppressors, that the arom gene cluster is transcribed, beginning with the arom-2 gene, as a single polycistronic messenger ribonucleic acid (mRNA) molecule which is subsequently translated into the arom multienzyme complex.     

Cold-sensitive Mutants of Neurospora crassa

Cold-sensitive mutants of the eucaryote Neurospora crassa have been isolated by a modification of the filtration-enrichment technique of Catcheside. The mutants include osmotic remedial, auxotrophic, transport, and incorporation deficient isolates.     

Suppressor Mutants of Neurospora Crassa That Tolerate Allelic Differences at Single or at Multiple Heterokaryon Incompatibility Loci

Allelic differences at any one of at least 11 heterokaryon incompatibility (het) loci in Neurospora crassa trigger an incompatibility response: localized cell death at sites of hyphal anastomosis. We have isolated spontaneous and insertional suppressor mutants that are heterokaryon-compatible in spite of allelic differences at one or at several het loci. Some intra- and extragenic mutants tolerated allelic differences only at single het loci. Multi-tolerant spontaneous mutants were isolated by selecting simultaneously for tolerance of differences at het-c, -d and -e, or at each of these plus mating-type. Some suppressor mutants were specific for only one allele at the affected het locus; others suppressed both alleles. Insertional mutations were isolated from banks of transformants, each having a plasmid integrated into a random position in the chromosome. One mutant tolerated allelic differences at het-d. A homologous cosmid from a Neurospora genomic bank complemented the mutant phenotype. A second insertional inactivation mutant was tolerant of het-c differences. Inactivation of the wild-type locus corresponding to the integration site was accomplished by repeat-induced point mutation (RIP). The RIP progeny, like the original mutant, were tolerant of differences at het-c. It may be possible to use such suppressor mutants as universal donors of hypovirulence in pathogenic fungi.     

Selection of Respiratory Mutants of Neurospora crassa

A method is described that permits the isolation of mutants that are defective in mitochondrial respiration. The techniques of inositol-less death and overlay with 2,3,5-triphenyl-2H-tetrazolium chloride are utilized to select for mutant colonies. Colonies that survive inositol-less death and fail to reduce the tetrazolium dye are then tested polarographically for cyanide-sensitive respiration. A preliminary characterization of three mutants obtained by this method is presented. The mutants have been characterized by their cyanide-sensitive respiration rate, growth rate, the state III respiration rate of isolated mitochondria, inhibition of the respiration of isolated mitochondria by cyanide and antimycin A, and cytochrome spectra. All of the mutants described differ from the parent strain in some of these aspects.     

The Murine Dilute Suppressor Gene Encodes a Cell Autonomous Suppressor

The murine dilute suppressor gene (dsu) suppresses the coat-color phenotype of three pigment mutations, dilute (d), ashen (ash) and leaden (ln), that each produce adendritic melanocytes. Suppression is due to the ability of dsu to partially restore (ash and ln), or almost completely restore (d), normal melanocyte morphology. While the ash and ln gene products have yet to be identified, the d gene encodes a novel myosin heavy chain (myosin 12), which is speculated to be necessary for the elaboration, maintenance, and/or function of melanocyte cell processes. To begin to discriminate between different models of dus action, we have produced aggregation chimeras between mice homozygous for dsu and mice homozygous for d to determine if dsu acts cell autonomously or cell nonautonomously. In addition, we have further refined the map location of dsu in order to examine a number of possible dsu candidate genes mapping in the region and to provide a genetic basis for the positional cloning of dsu.