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 .

Molecular machines, typically thought to be only the fanciful imaginings of speculative fiction, have taken great strides in recent years towards real-world viability and usefulness. Under variable temperature scanning tunneling microscopy, (STM) one family of these nascent devices is characterized with atomic resolution, and probed and manipulated with sub-angstrom precision, adding to the growing body of knowledge of how molecular devices behave and react at nanometer scales. Evidence of temperature-dependent rolling of wheel-like fullerene constituents on the Nanocar is discussed in light of newly developed image analysis techniques. Additionally, charge-transfer mediated behavior at step edges, both static and dynamic, is investigated on a Au(111) surface for a more complete understanding of translation and surface diffusion. Molecular flexibility is thought to aid in this three-dimensional atomic-step-crossing diffusion, and is explored and discussed across many species in the Nanocar family of molecules. In all, many similar molecules have been characterized and explored via STM with an eye towards their dynamic capabilities and surface behaviors, in the hopes that future, more complex versions can build on the nascent knowledge base beginning to be established here.

A method is described for the growth of high purity crystalline thin film C$\sb{60}$. Films grown by this method are analyzed by means of x-ray diffraction, low energy electron diffraction, and photoluminescence. An excimeric model of the processes leading to the observed photoluminescence spectrum is proposed. This model is supported by the locations of spectral features in the photoluminescence spectrum as well as by agreement with the cooling dependence, temperature dependence, and the lack of film thickness dependence of the photoluminescence spectrum. The model is used to explain the observed photoluminescence spectrum of polycrystalline C$\sb{60}$.

A flowing helium afterglow apparatus has been used to study thermal-energy electron exchange collisions between spin-polarized electrons and O$\sb2$ or NO molecules. Penning ionization of CO$\sb2$ by spin-polarized He(2$\sp3$S) metastable atoms is used to produce electrons which retain the spin orientation of the metastables, and which rapidly thermalize in the CO$\sb2$. The reactant gas (O$\sb2$ or NO), when introduced into the flowstream, causes a decrease in the electron spin-polarization. The electrons are then extracted from the flowtube for measurement of the polarization they retain. The rate constant for the reaction e$\sp{-}(\uparrow$) + X $\to$ e$\sp {-}(\downarrow$) + X can then be determined, given the amount of polarization decrease, reactant-gas density, and reaction time. The rates are found to be k(O$\sb2$) = (8 $\pm$ 3.5) x 10$\sp{-11}$ cm$\sp3$/sec and k(NO) = (9 $\pm$ 4) x 10$\sp{-11}$ cm$\sp3$/sec. An upper limit to the electron attachment rate for formation of an excited negative ion is derived from these measurements, and the contribution of exchange to the total scattering is discussed. In addition, a new, non-invasive technique for measuring electron-drift velocity in the flowtube is describe. Modifications of the afterglow apparatus and use of laser radiation for He(2$\sp3$S) spin-orientation enable it to produce an electron beam having moderate to high current and high spin polarization. Polarizations of 80% are achieved for currents up to 1$\mu$A, with 60% polarization retained at 25$\mu$A. This compares favorably with other polarized electron sources, making the afterglow apparatus a candidate for use as a beam source in high-energy electron accelerators.

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.

Single-walled carbon nanotubes have been produced in a continuous flow gasphase catalytic growth process, in a heated flow of carbon monoxide at pressures of 1--10 atmospheres and temperatures of 800--1200°C. Catalytic cluster nuclei for the nanotubes are generated in situ by thermal decomposition of iron pentacarbonyl. The yield of nanotubes and their diameter distribution can be varied by varying process parameters: nanotubes as small as 0.6 nm in diameter, corresponding to the size of a C60 molecule, have been generated. This process shows great promise for scaling up for bulk production of carbon nanotubes.

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.