Characterization of human glucocerebrosidase from different mutant alleles.

Human cDNA was mutagenized to duplicate six naturally occurring mutations in the gene for glucocere-brosidase. The mutant genes were expressed in NIH 3T3 cells. The abnormal human enzymes were purified by immunoaffinity chromatography and characterized. The Asn370----Ser mutant protein differed from normal enzyme in its inhibition by both conduritol B epoxide and glucosphingosine demonstrating that the 370 mutant enzyme has an abnormal catalytic site. In addition, the 370 mutant enzyme is less activated by saposin C, but more stimulated by phosphatidylserine than the wild type enzyme. The Arg463----Cys mutant protein was normal with respect to conduritol B epoxide and glucosphingosine inhibition, but was less activated by both saposin C and phosphatidylserine. The Arg120----Gln mutant protein was catalytically inactive. The Leu444----Pro, the pseudopattern, and the Pro415----Arg mutants appear to have reduced amounts of enzyme protein in cells. The studies demonstrated that mutations in the gene for glucocerebrosidase have different effects on the catalytic activity and stability of the enzyme.     

Two distinct Notch1 mutant alleles are involved in the induction of T-cell leukemia in c-myc transgenic mice

We have previously characterized a large panel of provirus insertion Notch1 mutant alleles and their products arising in thymomas of MMTV(D)/myc transgenic mice. Here, we show that these Notch1 mutations represent two clearly distinct classes. In the first class (type I), proviral integrations were clustered just upstream of sequences encoding the transmembrane domain. Type I Notch1 alleles produced two types of mutant Notch1 RNA, one of which encoded the entire Notch1 cytoplasmic domain [N(IC)] and the other of which encoded a soluble ectodomain [N(EC)(Mut)] which, in contrast to the processed wild-type ectodomain [N(EC)(WT)], did not reside at the cell surface and became secreted in a temperature-dependent manner. A second, novel class of mutant Notch1 allele (type II) encoded a Notch1 receptor with the C-terminal PEST motif deleted (DeltaCT). The type II Notch1(DeltaCT) protein was expressed as a normally processed receptor [N(EC)(WT) and N(IC)(DeltaCT)] at the cell surface, and its ectodomain was found to be shed into the extracellular medium in a temperature- and calcium-dependent manner. These data suggest that both type I and type II mutations generate two structurally distinct Notch1 N(EC) and N(IC) proteins that may participate in tumor formation, in collaboration with the c-myc oncogene, through distinct mechanisms. Constitutive type I N(IC) and type II N(IC)(DeltaCT) expression may enhance Notch1 intracellular signaling, while secreted or shed type I N(EC)(Mut) and type II N(EC) proteins may differentially interact in an autocrine or paracrine fashion with ligands of Notch1 and affect their signaling.     

Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1

The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.     

Expression of new mutant alleles of AS1 and AS2 genes controlling leaf morphogenesis in Arabidopsis thaliana

We have studied the morphology and vein branching of rosette leaves in Arabidopsis thaliana mutants as and sa, which proved to be alleles of the A.thaliana AS1 and AS2 genes, respectively. We have also analyzed the localization of bioactive auxin, as measured by the expression of the DR5::GUS transgene, as well as the expression patterns of BP, as measured by the expression of the BP::GUS transgene in leaves of the mutants. In mature leaves of the mutants, BP was expressed ectopically. Furthermore, the mutants showed some defects in the localization and concentration of free auxin compared to the wild type. Our results of studying new alleles of AS1 and AS2 support their role in control of class I KNOX genes and auxin transport.     

Expression of new mutant alleles of AS1 and AS2 genes controlling leaf morphogenesis in Arabidopsis thaliana

We have studied the morphology and vein branching of rosette leaves in Arabidopsis thaliana mutants as and sa, which proved to be alleles of the A. thaliana AS1 and AS2 genes, respectively. We have also analyzed the localization of bioactive auxin, as measured by the expression of the DR5::GUS transgene, as well as the expression patterns of BP, as measured by the expression of the BP::GUS transgene in leaves of the mutants. In mature leaves of the mutants, BP was expressed ectopically. Furthermore, the mutants showed some defects in the localization and concentration of free auxin compared to the wild type. Our results of studying new alleles of AS1 and AS2 support their role in control of class I KNOX genes and auxin transport.     

Characterization of a cytokinesis defective (cyd1) mutant of Arabidopsis.

Although several mutations and genes affecting plant cytokinesis have been identified, mutant screens are not yet saturated and knowledge about gene function is still limited. A novel Arabidopsis mutation, cytokinesis defective1 (cyd1), was identified by partial or missing cell walls in stomata. Stomata with incomplete or no cytokinesis still differentiate and some contain swellings of the outer wall not found in the wild type. The incomplete walls are correctly placed opposite stomatal wall thickenings suggesting that the mutation interferes with the execution of cytokinesis rather than with the placement of the division site. Cytokinesis defects are also detectable in other cell types throughout the plant, defects which include cell wall protrusions, two or more nuclei in one cell, and reduced cell number. The extent of cytokinetic partitioning correlates with nuclear number in abnormal stomata. Many cyd1 epidermal cells, stomata and pollen are larger, and trichomes have more branches. cyd1 is partially lethal with poor seed set and some defective ovules, but many plants are fertile despite abnormalities in vegetative and reproductive development such as missing, reduced, fused or misshapen leaves and floral organs. cyd1 appears to be the only cytokinesis mutant described where defects are known to occur in both mature vegetative and reproductive organs. Thus, the CYD1 gene product appears to be necessary for the execution of cytokinesis throughout the shoot. The examination of stomata by microscopy may be a useful screen for the directed isolation of additional cytokinesis mutations that are not embryo or seedling lethal     

Characterization of the Brassinosteroid Insensitive 1 Genes of Cotton

Suppression of brassinosteroid (BR) biosynthesis in cotton ovules by treatment with brassinazole inhibits fiber formation, indicating that BR plays an important role in cotton fiber development. Plant responses to brassinosteroids (BR) are mediated through a plasma membrane-bound leucine-rich repeat (LRR) receptor-like protein kinase known as BRI1. Mutations in the BRI1 genes of several species result in dwarfed plants with reduced sensitivity to BR. A single expressed sequence tag (EST) from cotton with strong sequence similarity to Arabidopsis BRI1 ( AtBRI1 ) was identified in a search of publicly available databases. With this EST as a starting point, full-length cDNAs and genomic coding sequences from upland cotton ( Gossypium hirsutum ) BRI1 ( GhBRI1 ) were obtained and characterized. Ectopic expression of this coding sequence in BR-insensitive Arabidopsis plants resulted in recovery of normal growth indicating that GhBRI1 is a functional homologue of AtBRI1. G. hirsutum is an allotetraploid (AADD) derived from diploid ancestors. Analysis of several GhBRI1 cDNAs showed two distinct sequences indicating the presence of two GhBRI1 genes, denoted GhBRI1-1 and GhBRI1-2. Sequence comparisons between these GhBRI1 coding sequences and those from related A and D genome diploid Gossypium species ( G. arboreum and G. thurberi ) indicated that GhBRI1-1 is likely to the A sub-genome orthologue while GhBRI1-2 is from the D sub-genome.     

Characterization of an Arabidopsis thaliana DNA hypomethylation mutant.

We have recently isolated two Arabidopsis thaliana DNA hypomethylation mutations, identifying the DDM1 locus, that cause a 70% reduction in genomic 5-methylcytosine levels [1]. Here we describe further phenotypic and biochemical characterization of the ddm1 mutants. ddm1/ddm1 homozygotes exhibited altered leaf shape, increased cauline leaf number, and a delay in the onset of flowering when compared to non-mutant siblings in a segregating population. Our biochemical characterization investigated two possible mechanisms for DNA hypomethylation. In order to see if ddm1 mutations affect DNA methyltransferase function, we compared DNA methyltransferase activities in extracts from wild-type and ddm1 mutant tissues. The ddm1 mutant extracts had as much DNA methyltransferase activity as that of the wild-type for both the CpI and CpNpG substrates suggesting that the DDM1 locus does not encode a DNA methyltransferase. Moreover, the ddm1 mutations did not affect the intracellular level of S-adenosylmethionine, the methyl group donor for DNA methylation. The possibility that the DDM1 gene product functions as a modifier of DNA methylation is discussed.     

Characterization of promoter activity of the ethylene receptor gene OgERS1 from Oncidesa in transgenic Arabidopsis

Physiological changes associated with senescence of flowers and abscission of floral parts in Oncidesa (formerly Oncidium) cv. Gower Ramsey are caused by a plant hormone ethylene which is produced by pollinia cap dislodgment during postharvest handling and transportation. The ethylene receptor gene OgERS1 of Oncidesa has been previously cloned and characterized. To analyze promoter activity of OgERS1, transgenic Arabidopsis thaliana plants were generated to express the ß-glucuronidase (GUS) reporter gene under the control of 5’-upstream sequence of OgERS1 from Oncidesa. The expression pattern of the OgERS1 promoter at the cellular level was investigated by analysis of GUS activity. This promoter can activate gene expression in both actively dividing young tissues and abscission-related aging tissues. Expression of GUS was detected in the shoot meristem uniquely in 10 to 30 d-old-plants and was found in flower buds, axillary buds, flower stems, and abscission layers during later development. In 2- to 3-week-old transgenic Arabidopsis, exogenous ethylene, glucose, lactose, and maltose enhanced promoter activity implying that crosstalk between sugar and an ethylene receptor may exist. However, indole-3-acetic acid, benzylaminopurine, abscisic acid, heat, wounding, salinity, drought, and flooding slightly suppressed promoter activity. These results demonstrate that the promoter of OgERS1 was developmentally and environmentally regulated, and imply a potential for application of this bi-functional promoter to increase branching or enhanced dwarfing.     

Characterization of waldmeister, a novel developmental mutant in Arabidopsis thaliana

A novel Arabidopsis thaliana (L.) Heynh. developmental mutant,waldmeister (wam), is described. This mutant was found in theprogeny arising from an Ac-Ds tagging experiment, but does notappear to be tagged by an introduced transposon. This recessivenuclear mutation maps between GAPB and ap1 on chromosome 1 andshows extreme morphological and physiological changes in bothfloral and vegetative tissues. Changes to the vegetative phenotypeinclude altered leaf morphology, multiple rosettes, stem fasciation,retarded senescence and disturbed geotropic growth. Changesto the floral phenotype include delayed flowering, increasednumber of inflorescences, determinate inflorescences, alterednumber and morphology of floral organs, chimeric floral organs,and ectopic ovules . wam was crossed to a number of previouslydescribed floral mutants: apetela 2, apetela 3, pistillata,agamous, and leafy. The phenotype of the double mutant was ineach case additive. In the case of agamous, however, the indeterminaterepetitive floral structure of agamous was lacking, emphasizingthe determinate inflorescence growth of wam. The extreme phenotypeof the wam mutant is suggestive of a disturbance to a gene ofglobal importance in the regulation of plant growth and development. Key words: Arabidopsis thaliana, waldmeister, developmental mutant, flower mutant     

Characterization of sunlight-grown transgenic rice plants expressing Arabidopsis phytochrome A

Transgenic rice (Oryza sativa) overexpressing Arabidopsis phytochrome A (phyA) was cultivated up to the T₃ generation in paddy to elucidate the role of phyA in determining the plant architecture and the productivity of sunlight-grown rice plants. PhyA is light-labile and controls plant growth in response to the far-red light-dependent high-irradiance response as well as the very low fluence response. The Arabidopsis phyA gene linked to the rice rbcS promoter was transformed into embryogenic rice calli, and the calli were regenerated to whole plants. Compared to wild-type seedlings, the rbcS::PHYA transgenic seedlings contained more phyA when grown in the dark, and at least 10-fold more phyA when exposed to white light. When grown in paddy, the phyA transgenic plants in general exhibited reduced plant height (dwarfing), larger grain size, higher chlorophyll content, smaller tiller number, and low grain fertility compared to wild-type plants. The heading stage was not significantly changed. However, it is likely that a certain level of phyA is a prerequisite for induction of such changes. It is suggested that phyA overproduction in rice could be a useful tool to improve rice grain productivity by the larger grain size that increases grain yield and the dwarfing that tolerates lodging-associated damage.     

Generation and maintenance of Dmbx1 gene-targeted mutant alleles

Dmbx1 encodes a paired-like homeodomain protein that is expressed in neural tissues at mouse embryonic and postnatal stages. We previously generated two Dmbx1 mutant alleles, Dmbx1 ⁻ and Dmbx1 ^z , by homologous recombination in mouse embryonic stem (ES) cells. In this article we report the generation of three novel Dmbx1 mutant alleles, Dmbx1 ^τZ , Dmbx1 ^τG , and Dmbx1 ^Cre , that carry the intronic insertion of tau (τ)-lacZ, τ-eGFP, and Cre reporter genes, respectively. Dmbx1 ^τZ and Dmbx1 ^τG recapitulated the Dmbx1 expression, and the reporter gene expression was detected in the diencephalon and mesencephalon during embryogenesis. The crossing of Dmbx1 ^Cre mice with Rosa26 reporter mice identified the Cre-mediated DNA excision in the postnatal midbrain, cerebellum, medulla oblongata, and spinal cord. To maintain the Dmbx1 mutant alleles without genotyping, we crossed Dmbx1 mutant mice with Inv4(1) ^Brd mice that possess the inversion between D4Mit117 and D4Mit281 on Chromosome 4, where Dmbx1 is located. The intercrossing of the non-agouti (a/a) albino (Tyr ^c-Brd /Tyr ^c-Brd ) Dmbx1 mutant mice carrying Inv4(1) ^Brd tagged with K14-Agouti and Tyrosinase coat-color markers resulted in the generation of dark brown Dmbx1 wild-type [Inv4(1) ^Brd /Inv4(1) ^Brd ], light brown Dmbx1 heterozygous [Dmbx1 ^tm /Inv4(1) ^Brd ], and albino Dmbx1 homozygous (Dmbx1 ^tm /Dmbx1 ^tm ) mutant mice. To our knowledge, this is the first demonstration of the proof-of-principle of the maintenance of viable gene-targeted alleles using coat-color-tagged nonlethal balancer chromosomes.     

Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance

In many plant-pathogen interactions, resistance is associated with the synthesis and accumulation of salicylic acid (SA) and pathogenesis-related (PR) proteins. At least two general classes of mutants with altered resistance to pathogen attack have been identified in Arabidopsis. One class exhibits increased susceptibility to pathogen infection; the other class exhibits enhanced resistance to pathogens. In an attempt to identify mutations in resistance-associated loci, we screened a population of T-DNA tagged Arabidopsis thaliana ecotype Wassilewskija (Ws) for mutants showing constitutive expression of the PR-1 gene (cep). A mutant was isolated and shown to constitutively express PR-1, PR-2, and PR-5 genes. This constitutive phenotype segregated as a single recessive trait in the Ws genetic background. The mutant also had elevated levels of SA, which are responsible for the cep phenotype. The cep mutant spontaneously formed hypersensitive response (HR)-like lesions on the leaves and cotyledons and also exhibited enhanced resistance to virulent bacterial and fungal pathogens. Genetic analyses of segregating progeny from outcrosses to other ecotypes unexpectedly revealed that alterations in more than one gene condition the constitutive expression of PR genes in the original mutant. One of the mutations, designated cpr20, maps to the lower arm of chromosome 4 and is required for the cep phenotype. Another mutation, which has been termed cpr21, maps to chromosome 1 and is often, but not always, associated with this phenotype. The recessive nature of the cep trait suggests that the CPR20 and CPR21 proteins may act as negative regulators in the disease resistance signal transduction pathway.