We have defined amino acids important for function of the Arabidopsis thaliana Hsp100/ClpB chaperone (AtHsp101) in acquired thermotolerance by isolating recessive, loss-of-function mutations and a novel semidominant, gain-of-function allele [hot1-4 (A499T)]. The hot1-4 allele is unusual in that it not only fails to develop thermotolerance to 458C after acclimation at 388C, but also is sensitive to 388C, which is a permissive temperature for wild-type and loss-of-function mutants. hot1-4 lies between nucleotide binding domain 1 (NBD1) and NBD2 in a coiled-coil domain that is characteristic of the Hsp100/ClpB proteins. We then isolated two classes of intragenic suppressor mutations of hot1-4: loss-of-function mutations (Class 1) that eliminated the 388C sensitivity, but did not restore thermotolerance function to hot1-4, and Class 2 suppressors that restored acquired thermotolerance function to hot1-4. Location of the hot1-4 Class 2 suppressors supports a functional link between the coiled-coil domain and both NBD1 and the axial channel of the Hsp100/ClpB hexamer. In addition, the strongest Class 2 suppressors restored solubility of aggregated small heat shock proteins (sHsps) after heat stress, revealing genetic interaction of the Hsp100/ClpB and sHsp chaperone systems. These results also demonstrate that quantitative phenotypes can be used for in vivo genetic dissection of protein mechanism in Arabidopsis.

Subtle cellular phenotypes in the CNS may evade detection by routine histopathology. Here, we demonstrate the value of primary culture for revealing genetically determined neuronal phenotypes at high resolution. Gamma neurons of Drosophila melanogaster mushroom bodies (MBs) are remodeled during metamorphosis under the control of the steroid hormone 20-hydroxyecdysone (20E). In vitro, wild-type ␥ neurons retain characteristic morphogenetic features, notably a single axon-like dominant primary process and an arbor of short dendrite-like processes, as determined with microtubule-polarity markers. We found three distinct genetically determined phenotypes of cultured neurons from grossly normal brains, suggesting that subtle in vivo attributes are unmasked and amplified in vitro. First, the neurite outgrowth response to 20E is sexually dimorphic, being much greater in female than in male ␥ neurons. Second, the ␥ neuron-specific "naked runt" phenotype results from transgenic insertion of an MB-specific promoter. Third, the recessive, panneuronal "filagree" phenotype maps to singed, which encodes the actin-bundling protein fascin. Fascin deficiency does not impair the 20E response, but neurites fail to maintain their normal, nearly straight trajectory, instead forming curls and hooks. This is accompanied by abnormally distributed filamentous actin. This is the first demonstration of fascin function in neuronal morphogenesis. Our findings, along with the regulation of human Fascin1 (OMIM 602689) by CREB (cAMP response element-binding protein) binding protein, suggest FSCN1 as a candidate gene for developmental brain disorders. We developed an automated method of computing neurite curvature and classifying neurons based on curvature phenotype. This will facilitate detection of genetic and pharmacological modifiers of neuronal defects resulting from fascin deficiency.

To evaluate the genetic control of stress responses in Arabidopsis, we have analyzed a mutant (uvh6-1) that exhibits increased sensitivity to UV light, a yellow-green leaf coloration, and mild growth defects. We have mapped the uvh6-1 locus to chromosome I and have identified a candidate gene, AtXPD, within the corresponding region. This gene shows sequence similarity to the human (Homo sapiens) XPD and yeast (Saccharomyces cerevisiae) RAD3 genes required for nucleotide excision repair. We propose that UVH6 is equivalent to AtXPD because uvh6-1 mutants carry a mutation in a conserved residue of AtXPD and because transformation of uvh6-1 mutants with wild-type AtXPD DNA suppresses both UV sensitivity and other defective phenotypes. Furthermore, the UVH6/AtXPD protein appears to play a role in repair of UV photoproducts because the uvh6-1 mutant exhibits a moderate defect in the excision of UV photoproducts. This defect is also suppressed by transformation with UVH6/AtXPD DNA. We have further identified a T-DNA insertion in the UVH6/AtXPD gene (uvh6-2). Plants carrying homozygous insertions were not detected in analyses of progeny from plants heterozygous for the insertion. Thus, homozygous insertions appear to be lethal. We conclude that the UVH6/AtXPD gene is required for UV resistance and is an essential gene in Arabidopsis.DNA damage is a challenge for all organisms exposed to UV irradiation. UV photoproducts consist primarily of cyclobutane pyrimidine dimers and pyrimidine (6-4) pyrimidinone dimers (Mitchell and Nairn, 1989;Pfeifer, 1997). These lesions inhibit DNA replication and transcription and also promote mutagenesis (McGregor, 1999). The effects of UV irradiation are especially detrimental in plants, where sunlight is both a source of damage and a requirement for photosynthesis.Increasing evidence suggests that plants repair UVdamaged chromosomes using mechanisms similar to those found in humans (Homo sapiens) and yeast (Saccharomyces cerevisiae). These mechanisms include the nucleotide excision repair (NER) pathway, a process which involves recognition of UV lesions, incision of the damaged strand on both sides of the lesion, removal of the damaged fragment, and repair by gap filling and ligation (Batty and Wood, 2000;de Boer and Hoeijmakers, 2000;Prakash and Prakash, 2000). Several potential plant homologs of human and yeast NER genes have been identified. Genetic analyses of these plant genes, including studies of the phenotypes of plants carrying mutations within these genes, provide support for the idea that the NER pathway is conserved in plants.Lesion recognition during NER involves the homologous heterodimers XPC:HR23B (human) and RAD4:RAD23 (yeast; Balajee and Bohr, 2000;Batty and Wood, 2000;de Boer and Hoeijmakers, 2000;Prakash and Prakash, 2000). The Arabidopsis genome contains potential homologs of both XPC/RAD4 and HR23B/RAD23 (Arabidopsis Genome Initiative, 2000). HR23B expression occurs in Arabidopsis, rice (Oryza sativa), and carrot (Daucus carota), and the carrot gene complements the r...