Role of aromatic residues in stabilization of the [Fe4S4] cluster in high-potential iron proteins (HiPIPs): physical characterization and stability studies of Tyr-19 mutants of Chromatium vinosum HiPIP.

The functional role of residue Tyr-19 of Chromatium vinosum HiPIP has been evaluated by site-directed mutagenesis experiments. The stability of the [Fe4S4] cluster prosthetic center is sensitive to side-chain replacements. Polar residues result in significant instability, while nonpolar residues (especially with aromatic side chains) maintain cluster stability. Two-dimensional NMR data of native and mutant HiPIPs are consistent with a model where Tyr-19 serves to preserve the structural rigidity of the polypeptide backbone, thereby maintaining a hydrophobic barrier for exclusion of water from the cluster cavity. Solvent accessibility results in more facile oxidation of the cluster by atmospheric oxygen, with subsequent rapid hydrolysis of the [Fe4S4]3+ core.

Targeted gene inactivation in petunia by PCR-based selection of transposon insertion mutants.

Establishment of loss-of-function phenotypes is often a key step in determining the biological function of a gene. We describe a procedure to obtain mutant petunia plants in which a specific gene with known sequence is inactivated by the transposable element dTph1. Leaves are collected from batches of 1000 plants with highly active dTph1 elements, pooled according to a three-dimensional matrix, and screened by PCR using a transposon- and a gene-specific primer. In this way individual plants with a dTph1 insertion can be identified by analysis of about 30 PCRs. We found insertion alleles for various genes at a frequency of about 1 in 1000 plants. The plant population can be preserved by selfing all the plants, so that it can be screened for insertions in many genes over a prolonged period.

Benzodiazepine-insensitive mice generated by targeted disruption of the gamma 2 subunit gene of gamma-aminobutyric acid type A receptors.

Vigilance, anxiety, epileptic activity, and muscle tone can be modulated by drugs acting at the benzodiazepine (BZ) site of gamma-aminobutyric acid type A (GABAA) receptors. In vivo, BZ sites are potential targets for endogenous ligands regulating the corresponding central nervous system states. To assess the physiological relevance of BZ sites, mice were generated containing GABAA receptors devoid of BZ sites. Following targeted disruption of the gamma 2 subunit gene, 94% of the BZ sites were absent in brain of neonatal mice, while the number of GABA sites was only slightly reduced. Except for the gamma 2 subunit, the level of expression and the regional and cellular distribution of the major GABAA receptor subunits were unaltered. The single channel main conductance level and the Hill coefficient were reduced to values consistent with recombinant GABAA receptors composed of alpha and beta subunits. The GABA response was potentiated by pentobarbital but not by flunitrazepam. Diazepam was inactive behaviorally. Thus, the gamma 2 subunit is dispensable for the assembly of functional GABAA receptors but is required for normal channel conductance and the formation of BZ sites in vivo. BZ sites are not essential for embryonic development, as suggested by the normal body weight and histology of newborn mice. Postnatally, however, the reduced GABAA receptor function is associated with retarded growth, sensorimotor dysfunction, and drastically reduced life-span. The lack of postnatal GABAA receptor regulation by endogenous ligands of BZ sites might contribute to this phenotype.

Adaptation of Spodoptera exigua larvae to plant proteinase inhibitors by induction of gut proteinase activity insensitive to inhibition.

Tobacco plants were transformed with a cDNA clone of chymotrypsin/trypsin-specific potato proteinase inhibitor II (PI2) under the control of a constitutive promoter. Although considerable levels of transgene expression could be demonstrated, the growth of Spodoptera exigua larvae fed with detached leaves of PI2-expressing plants was not affected. Analysis of the composition of tryptic gut activity demonstrated that only 18% of the proteinase activity of insects reared on these transgenic plants was sensitive to inhibition by PI2, whereas 78% was sensitive in insects reared on control plants. Larvae had compensated for this loss of tryptic activity by a 2.5-fold induction of new activity that was insensitive to inhibition by PI2. PI2-insensitive proteolytic activity was also induced in response to endogenous proteinase inhibitors of tobacco; therefore, induction of such proteinase activity may represent the mechanism by which insects that feed on plants overcome plant proteinase inhibitor defense.

SCT1 mutants suppress the camptothecin sensitivity of yeast cells expressing wild-type DNA topoisomerase I.

Camptothecin is a potent antineoplastic agent that interferes with the action of eukaryotic DNA topoisomerase I; the covalent enzyme-DNA intermediate is reversibly stabilized, leading to G2 arrest and cell death. We used a genetic screen to identify cellular factors, other than DNA topoisomerase I, that participate in the process of camptothecin-induced cell death. Following ethyl methanesulfonate mutagenesis of top1 delta yeast cells expressing plasmid-borne wild-type DNA topoisomerase I, six dominant suppressors of camptothecin toxicity were isolated that define a single genetic locus, sct1. Mutant SCT1 cells expressed DNA topoisomerase I protein of similar specific activity and camptothecin sensitivity to that of congenic, drug-sensitive sct1 cells, yet were resistant to camptothecin-mediated lethality. Moreover, camptothecin-treated SCT1 cells did not exhibit the G2-arrested, terminal phenotype characteristic of drug-treated wild-type cells. SCT1 cell sensitivity to other DNA-damaging agents suggests that alterations in SCT1 function suppress camptothecin-induced DNA damage produced in the presence of yeast DNA topoisomerase I.

A chimeric light-regulated amino acid transport system allows the isolation of blue light regulator (blr) mutants of Neurospora crassa.

We have developed a system for the isolation of Neurospora crassa mutants that shows altered responses to blue light. To this end we have used the light-regulated promoter of the albino-3 gene fused to the neutral amino acid permease gene mtr. The product of the mtr gene is required for the uptake of neutral aliphatic and aromatic amino acids, as well as toxic analogs such as p-flurophenylalanine or 4-methyltryptophan. mtr trp-2-carrying cells were transformed with the al-3 promoter-mtr wild-type gene (al-3p-mtr+) to obtain a strain with a light-regulated tryptophan uptake. This strain is sensitive to p-fluorophenylalanine when grown under illumination and resistant when grown in the dark. UV mutagenesis of the al-3p-mtr(+)-carrying strain allowed us to isolate two mutant strains, BLR-1 and BLR-2 (blue light regulator), that are light-resistant to p-fluorophenylalanine and have lost the ability to grow on tryptophan. These two strains have a pale-orange phenotype and show down-regulation of all the photoregulated genes tested (al-3, al-1, con-8, and con-10). Mutations in the BLR strains are not allelic with white collar 1 or white collar 2, regulatory genes that are also involved in the response to blue light.

A cytoplasmically transmissible hypovirulence phenotype associated with mitochondrial DNA mutations in the chestnut blight fungus Cryphonectria parasitica.

Mutations causing mitochondrial defects were induced in a virulent strain of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr. Virulence on apples and chestnut trees was reduced in four of six extensively characterized mutants. Relative to the virulent progenitor, the attenuated mutants had reduced growth rates, abnormal colony morphologies, and few asexual spores, and they resembled virus-infected strains. The respiratory defects and attenuated virulence phenotypes (hypovirulence) were transmitted from two mutants to a virulent strain by hyphal contact. The infectious transmission of hypovirulence occurred independently of the transfer of nuclei, did not involve a virus, and dynamically reflects fungal diseases caused by mitochondrial mutations. In these mutants, mitochondrial mutations are further implicated in generation of the attenuated state by (i) uniparental (maternal) inheritance of the trait, (ii) presence of high levels of cyanide-insensitive mitochondrial alternative oxidase activity, (iii) cytochrome deficiencies, and (iv) structural abnormalities in the mtDNA. Hence, cytoplasmically transmissible hypovirulence phenotypes found in virus-free strains of C. parasitica from recovering trees may be caused by mutant forms of mtDNA.

Use of yeast artificial chromosomes (YACs) in studies of mammalian development: production of beta-globin locus YAC mice carrying human globin developmental mutants.

To test whether yeast artificial chromosomes (YACs) can be used in the investigation of mammalian development, we analyzed the phenotypes of transgenic mice carrying two types of beta-globin locus YAC developmental mutants: (i) mice carrying a G-->A transition at position -117 of the A gamma gene, which is responsible for the Greek A gamma form of hereditary persistence of fetal hemoglobin (HPFH), and (ii) beta-globin locus YAC transgenic lines carrying delta- and beta-globin gene deletions with 5' breakpoints similar to those of deletional HPFH and delta beta-thalassemia syndromes. The mice carrying the -117 A gamma G-->A mutation displayed a delayed gamma- to beta-globin gene switch and continued to express A gamma-globin chains in the adult stage of development as expected for carriers of Greek HPFH, indicating that the YAC/transgenic mouse system allows the analysis of the developmental role of cis-acting motifs. The analysis of mice carrying 3' deletions first provided evidence in support of the hypothesis that imported enhancers are responsible for the phenotypes of deletional HPFH and second indicated that autonomous silencing is the primary mechanism for turning off the gamma-globin genes in the adult. Collectively, our results suggest that transgenic mice carrying YAC mutations provide a useful model for the analysis of the control of gene expression during development.

Use of Arabidopsis thaliana defense-related mutants to dissect the plant response to pathogens.

The plant defense response to microbial pathogens had been studied primarily by using biochemical and physiological techniques. Recently, several laboratories have developed a variety of pathosystems utilizing Arabidopsis thaliana as a model host so that genetic analysis could also be used to study plant defense responses. Utilizing a pathosystem that involves the infection of Arabidopsis with pathogenic pseudomonads, we have cloned the Arabidopsis disease-resistance gene RPS2, which corresponds to the avirulence gene avrRpt2 in a gene-for-gene relationship. RPS2 encodes a 105-kDa protein containing a leucine zipper, a nucleotide binding site, and 14 imperfect leucine-rich repeats. The RPS2 protein is remarkably similar to the product of the tobacco N gene, which confers resistance to tobacco mosaic virus. We have also isolated a series of Arabidopsis mutants that synthesize decreased levels of an Arabidopsis phytoalexin called camalexin. Analysis of these mutants indicated that camalexin does not play a significant role in limiting growth of avirulent Pseudomonas syringae strains during the hypersensitive defense response but that it may play a role in limiting the growth of virulent strains. More generally, we have shown that we can utilize Arabidopsis to systematically dissect the defense response by isolation and characterization of appropriate defense-related mutants.

Immunolocalization of the mercurial-insensitive water channel and glycerol intrinsic protein in epithelial cell plasma membranes.

Two water channel homologs were cloned recently from rat kidney, mercurial-insensitive water channel (MIWC) and glycerol intrinsic protein (GLIP). Polyclonal antibodies were raised against synthetic C-terminal peptides and purified by affinity chromatography. MIWC and GLIP antibodies recognized proteins in rat kidney with an apparent molecular mass of 30 and 27 kDa, respectively, and did not cross-react. By immunofluorescence, MIWC and GLIP were expressed together on the basolateral plasma membrane of collecting duct principal cells in kidney. By immunohistochemistry, MIWC and GLIP were expressed on tracheal epithelial cells with greater expression of GLIP on the basal plasma membrane and MIWC on the lateral membrane; only MIWC was expressed in bronchial epithelia. In eye, GLIP was expressed in conjunctival epithelium, whereas MIWC was found in iris, ciliary body, and neural cell layers in retina. MIWC and GLIP colocalized on the basolateral membrane of villus epithelial cells in colon and brain ependymal cells. Expression of MIWC and GLIP was not detected in small intestine, liver, spleen, endothelia, and cells that express water channels CHIP28 or WCH-CD. These studies suggest water/solute transporting roles for MIWC and GLIP in the urinary concentrating mechanism, cerebrospinal fluid absorption, ocular fluid balance, fecal dehydration, and airway humidification. The unexpected membrane colocalization of MIWC and GLIP in several tissues suggests an interaction at the molecular and/or functional levels.