I have identified regulatory elements in the pathogen Candida albicans that enable response to nitrosative stress. This dimorphic fungus typically resides in the digestive and genitourinary tracts as an innocuous constituent of the normal microflora but can opportunistically cause superficial mucosal infection. In immunocompromised patients, such infections may also progress to potentially lethal systemic disease. One adaptation that facilitates survival of C. albicans against the hostile environment inside the mammalian body is the ability to resist toxic reactive nitrogen species (RNS) generated by macrophages of the host immune system. Recent studies have shown that exposing C. albicans to nitric oxide, one type of RNS, induces upregulation of the flavohemoglobin Yhb1p. This protein confers protection by enzymatically converting nitric oxide to harmless nitrate, but it is unknown how C. albicans is able to detect nitric oxide in its environment and thus initiate this defense only as needed. I therefore analyzed this problem by incrementally mutating the YHB1 regulatory region to identify a nitric oxide-responsive element (NORE) that is required for NO sensitivity. Five transcription factor candidates of the Zn(II)2-Cys6 family were then isolated by using magnetic beads coated with this DNA element in crude whole cell extracts. Of the five, only deletion of the CTA4 gene prevented induction of YHB1 transcription during nitrosative stress and caused growth sensitivity to the nitric oxide donor DPTA NONOate. The virulence of the cta4Delta deletion mutant was also mildly impaired, slightly more so than that of a yhb1Delta deletion mutant. Cta4p is the first protein found to be necessary for nitric oxide response in C. albicans .

A 550 bp DNA fragment which apparently contains part of the rubredoxin oxidoreductase gene has been amplified by a polymerase chain reaction employing genonic DNA as template. Oligonucleotides used in this amplification were designed based on the multiple alignment of rubredoxin oxidoreductase from related species. An EMBL3 $\lambda$ phage library of C. acetobutylicum genomic DNA was screened by an oligonucleotide hybridization method. The phages which hybridized with the radiolabeled PCR product were subcloned into pUC19 and pACYC184 vectors, with the recombinant plasmids being selected on the basis of white/blue color screening method and the insertional inactivation method, respectively. The probable identity of the pUC19 clone, which was designated RuNC51 and carried a 3.0 kb BglII fragment, was confirmed by rubredoxin oxidoreductase enzyme activity assay. The sequence of RuNC51 showed the same level of similarity to related genes. It also surprisingly showed a 53% similarity to PriA (protein n$\sp\prime$) which is involved in DNA replication.

The typical eukaryotic RNA polymerase II primary transcript is divided into regions that encode information expressed at the protein level (exons) and those which do not (introns). The latter must be removed from the transcript rapidly and with proper joining of the coding sequences during the maturation of the transcript in the nucleus. This process is termed splicing and is accompanied by the sequential addition of factors to the primary transcript resulting in the formation of a series of large ribonucleoprotein particles. The splicing reaction can be studied in vitro in HeLa cell nuclear extracts by the addition of a capped, in vitro transcribed splicing precursor RNA. A native gel electrophoresis system was developed which allowed resolution of various ribonucleoprotein complexes and the study of the splicing complex and intermediates in its formation. The HeLa cell nuclear extracts were found to immediately assemble exogenously added precursor RNAs into rapidly migrating complexes. With time, complexes migrating more slowly were observed. The complex migrating the most slowly appeared concurrently with the products of 5$\sp\prime$ junction cleavage. This complex was identified as the "active" splicing complex by the presence of reaction intermediates and the requirement for both ATP and splicing consensus sequences for its formation. Later in the reation, when large amounts of ligated RNA had been generated, rapidly migrating complexes reappeared. All of these complexes contained U snRNPs as defined by immunoprecipitation of gel-fractionated complexes. In particular, eluted active complex contained U1 snRNPs as defined by the ability of anti-U1 antiserum to immunoprecipitate this particle in the presence of competing free, unlabelled precursor RNA. Previously reported gel systems appear to resolve active complexes devoid of U1 snRNPs, a snRNP known to be required for splicing both in vivo and in vitro. Furthermore, the 5$\sp\prime$ splice junction, the region to which U1 snRNPs bind, was protected from oligonucleotide directed RNase H cleavage in eluted complexes, indicating that a 5$\sp\prime$ factor remained bound during electrophoresis. Apparently, complexes eluted from the native system retain most of the properties, other than activity, found for these complexes in whole extracts, suggesting that this gel system is an ideal tool for the study of the ribonucleoprotein complexes involved in splicing.

The HOG MAP kinase pathway in the budding yeast Saccharomyces cerevisiae senses and responds to high osmolarity. Here we demonstrated that HOG pathway mutants are hypersensitive to K1 killer toxin, which implies certain defects in their cell wall. Overexpression of the PBS2 gene leads to enhanced resistance to K1 killer toxin. Treating yeast cells with a pore-forming antifungal agent, amphotericin B, lowers the cellular turgor pressure. More importantly, amphotericin B treatment leads to activation of the HOG pathway, supporting the hypothesis that loss of turgor pressure activates the HOG pathway. Deficiencies in the HOG pathway also cause hypersensitivity to hydrogen peroxide and the superoxide-generating drug plumbagin. Hydrogen peroxide, menadione and plumbagin all activate the HOG pathway. The HOG pathway acts parallel to Skn7p and Yap1p in oxidative stress response, evidenced by the additive effect of hog1Delta, skn7Delta and yap1Delta on hydrogen peroxide sensitivity. Both ssn6Delta and sko1Delta suppress hog1Delta mutant sensitivity to oxidants. Oxidative stress induces transcription of HSP12 and HSP26. The HOG pathway regulates HSP12 transcription in this response. Msn2p and Msn4p are important for the oxidative stress-induced transcription of HSP12 and HSP26. The HOG pathway is also involved in heat shock response. Cells lacking the HOG1 gene are hypersensitive to heat stress. A temperature shift from 25°C to 37°C activates the HOG pathway. Such an increase in temperature also induces transcription of HSP12 and HSP26. The HOG pathway regulates HSP12 transcription in the heat shock response. Msn2p and Msn4p are important for the heat shock-induced transcription of HSP12 and HSP26.

We have identified the lmo4 locus as a regulator of the size and organization of the eye. Using the yeast 2 hybrid system, we have identified and characterized Lmo4-Ldb interactions. We show that Lmo4 interacts with zebrafish Ldb1, Ldb2, and Ldb3 (LIM-domain binding) proteins, albeit more strongly with Ldb1. This interaction can be mediated by both LIM domains of Lmo4, though the interaction is much stronger with LIM B rather than LIM A. Mutating both LIM domains through strategic point mutations of key zinc coordinating residues abolishes their ability to interact with Ldbs. Mutant overexpression studies showed that overexpressing LIM B results in a mild small eye phenotype whereas overexpressing LIM A does not. Furthermore, Ldb1, Ldb2, and Ldb3 can heterodimerize and all can interact with zebrafish Islet3, which belongs to the LIM-homeodomain class of proteins and is suggested by others to play an essential role in eye development. We have tested the model of islet3 antagonism by lmo4 through morpholino knock down of islet3 and attempted rescue of the lmo4 gain of function phenotype by islet3 overexpression. Knock down of islet3 does not result in an eye phenotype, and rescue experiments show that the lmo4 overexpression phenotype can not be rescued by ectopic isl3 overexpression. We conclude that islet3 is not necessary for eye development in zebrafish, and the role of lmo4 with respect to eye development must be mediated through an antagonism of other lhx genes or through an lhx-independent mechanism.

Sugars serve as signaling molecules in plants, affecting gene expression and a number of developmental processes, but their precise role and the pathways through which they act are not well characterized. In addition, sugar-signaling pathways interact with the vast network of phytohormone-signaling pathways. In order to facilitate an understanding of sugar sensing and signaling pathways in Arabidopsis, sugar-insensitive mutants have been isolated for study and global genome analysis of wild-type and mutant responses to sugars and the phytohormones abscisic acid, ethylene, and gibberellin have been undertaken. Sugar-insensitive mutants were isolated based on their ability to form true leaves and expanded cotyledons in the presence of high concentrations (0.27 M to 0.34 M) of sucrose or glucose. The sis3 and sis6 mutants were chosen for further study. The sis3-1 mutation is recessive and is not linked to the presence of a T-DNA insert carried by the mutant. The sis3 mutation maps to the bottom arm of chromosome 3. The sis3 mutant is slightly insensitive to the effects of exogenously applied abscisic acid (ABA) on seed germination and root elongation but has a wild-type response to all other phytohormones tested. The sis6-1 mutant carries a cDNA encoding the At4g28240 putative wound-inducible gene on a T-DNA insert. The dominant nature of the mutation suggests that the five-fold overexpression of the cDNA in the mutant may be the cause of the sis phenotype. However, disruption of a gene by the T-DNA insertion is also a possible cause of the phenotype. The sis6 mutant is resistant to the inhibition of seed germination and root elongation caused by application of exogenous ABA. It is also resistant to the inhibition of germination caused by application of exogenous paclobutrazol (an inhibitor of gibberellin biosynthesis). This suggests a possible link between sugar and gibberellin signaling pathways. Analysis of gene expression in response to sugars and phytohormones was undertaken using the Affymetrix GeneChip Arabidopsis ATH 1 Genome Array which includes over 24,000 Arabidopsis genes. Both wild-type germinating seeds and adult plants were studied in order to begin determining the impact of the developmental age of the plant on sugar-regulated gene expression.

We present evidence that magainin I, a 23 residue antibiotic peptide, undergoes a concentration dependent phase transition in synthetic lipid multilayers. Using oriented circular dichroism (OCD) we show that in low concentrations, magainin forms $\alpha$-helices lying parallel to the membrane surface. When the concentration becomes greater than a lipid specific value, the magainin undergoes a conformational change to a yet unidentified state. The state of the system at concentrations above the critical concentration shows that this transition cannot be explained by the micellar effect. Correlations between our results and liposome leakage experiments indicate that this transition is responsible for magainin's antibiotic action.

This research has investigated the effect that altered supercoiling has on in vivo expression from bacterial promoters. In vivo expression was measured using the pKO system developed by McKenney et al. (1982). Supercoil levels were decreased through the use of the antibiotic nalidixic acid and were increased using a topoisomerase I deletion mutant. Galactokinase expression from a series of related hybrid promoters, the trp promoter flanked by oligonucleotide blocks, the trp promoter flanked by linker sections and the trp promoter flanked by a gyrase binding site was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter was inhibited by 50% under these conditions. Expression from the lacUV5, trp, and tettrp promoters was essentially unaffected. A 2-fold stimulation of expression was observed from the trplac and the trptet promoters. Expression from the trp promoter when flanked by upstream or downstream oligonucleotide blocks was similar to the values observed from control plasmids. The presence of a gyrase binding site (Kirkegaard and Wang, 1981) also exerted little influence on overall expression after a decrease in supercoiling. To further study control of expression from the trp promoter, a series of plasmids was constructed which contained a synthetic lac operator at defined positions either upstream or downstream of the promoter. Downstream lac repressor binding diminished the levels of expression, while upstream binding had little effect on expression. Placement of the lac operator farther downstream decreased the level of repression observed. Plasmids were also constructed containing two lac operators, one upstream at -39 or -52 and an additional operator downstream. Galactokinase expression from these plasmids was decreased more than in those plasmids with only one lac operator. In addition a system was developed that combined the M13 bacteriophage system of Messing (1981) and the pKO system of McKenney et al. (1982) in order to measure short term changes in transcription. Gene fragments from the (beta)-lactamase gene of pBR322 and the galactokinase gene of pKO-1 were cloned into M13mp8. Single strand DNA from these constructions will hybridize with mRNA transcribed from pKO derivatives, allowing measurement of transient changes in transcription.

The available cloned examples of the intron-28S coding sequence junctions from the rDNA of Calliphora erythrocephala have been sequenced. These introns interrupt the rDNA at the same position as the type 1 intron family first detected in D. melanogaster and D. virilis. A duplication of 14 bp of the 28S rRNA coding sequence surrounds a shortened 2.8 kb version of the major genomic length class of introns. This duplication is characteristic of the type 1 rDNA intron family in D. melanogaster and D virilis. Comparison of the intron sequences from the three Dipteran flies has revealed considerable homology between the 3' intron sequences of C. erythrocephala and D. virilis. The 28S coding sequences are highly conserved between these flies. However, a region of diverence has been located and found to correspond to the eukaryotic analogue of the L1 ribosomal protein binding site of the prokaryotic 23S rRNA. A set of overlapping phage clones representing 20.5 kb of the region surrounding the calmodulin gene of D. melanogaster has been isolated from a genomic library. Mapping of the protein coding regions suggests that the calmodulin gene is divided into four exons and three introns. Intron A has separated the initiation codon from the coding regions. Introns B and C, beween amino acids 58 and 59 and amino acids 139 and 140, respectively, split two of the four calcium binding domains. The amino acid sequence of residues 1 through 139 of calmodulin has been deduced from the DNA sequence. In comparison to the mammalian calmodulin sequences, there is a single amino acid substitution at position 99, where phenylalanine replaces tyrosine. A comparison of the D. melanogaster calmodulin gene sequences to other known calmodulin genes shows from 79% to 85% homology.

The genomic organization and sequence of the 5S RNA genes of the Dipteran fly Calliphora erythrocephala have been determined. The 5S RNA genes consist of a tandem array of 480 base pair (bp) repeating units which are clustered at a single locus 4b on chromosome 2. The repeating units show only minimal sequences and length heterogeneity, and a consensus sequence for a cloned series of 5S repeat units was determined. The coding sequence for the mature 5S RNA contains a single residue change from the known gene sequences from three Drosophila species (19,30). A precursor RNA containing additional 3' sequences with some homology to the equivalent sequences of the Drosophila species (19,30) is indicated. Partial pseudogenes homologous to the extreme 3' end of the transcribed region and the adjacent termination sequence are found at two positions in the spacer. Comparison of the 5S RNA genes of C. erythrocephala to those of three Drosophila species (19,39) identified a striking series of perfectly conserved homologies identically positioned ((+OR-)1 nucleotide) within the 5' flanking DNA of all four Dipteran 5S RNA coding regions. One of the Dipteran homology blocks is highly conserved in sequence and position in all but one of the eukaryotic 5S RNA gene sequences examined (17/18 genes). A cloned library of genomic DNA sequences of C. erythrocephala was prepared and screened by differential hybridization to ('32)P-cDNA made against poly(A)('+) RNA from embryos and two stages of oogenesis to detect sequences specifically transcribed during oogenesis. Four phage clones showed transcriptional activity in specific stages of oogenesis but not during embryogenesis. Each clone hybridized to a single poly(A)('+) RNA transcript which differed in size and stage-specificity for the four clones. All four clones contain DNA sequences which are highly repeated within the C. erythrocephala genome, but the transcriptional activity, if any, of the repetitive elements in each clone is undetermined. Transcribed fragments from each clone failed to hybridize to D. melanogaster genomic DNA at hybridization conditions allowing 10% sequence divergence. Whole follicles were used in the identification of these oogenesis-specific phage clones, and the cell specificity (follicle cell or nurse cell) is, as yet, undetermined.