Telomerase is the major mechanism that compensates for telomere loss during conventional DNA replication in eukaryotic cells. This enzyme is a ribonucleoprotein reverse transcriptase, composed of an RNA subunit (TR) containing a template that dictates the telomeric DNA sequence, a catalytic subunit (TERT), and others associated proteins. In order to characterize the telomerase RNA-proteins interactions in two different organisms, Tetrahymena thermophila and human, we developed two electrophoretic RNA mobility shift assays. In Tetrahymena, we identified a specific RNA-protein complex by using whole cell extracts partially purified and in vitro transcribed and radiolabeled Tetrahymena telomerase RNA (159 nt). Certain Tetrahymena telomerase RNA mutants tested in competition with wild type RNA by shift assays and containing deletions of structures and sequences previously predicted to be involved in protein binding were unable to competitively inhibit complex formation, suggesting a role in protein binding for the deleted residues or structures. In human, we identified a specific telomerase RNA-protein complex by using 293 human cell extracts partially purified, and in vitro transcribed and radiolabeled hTR (451 nt). Using different hTR mutants in competition with wild type hTR, we identified nucleotides 1--424 of hTR as a minimal region that is able to reconstitute a stable RNA-protein complex.

Increasing industrial applications of selenium as well as its use in supplementation studies at elevated but sublethal concentrations warrant further investigation into the mechanism(s) of selenium toxicity. Through use of an Escherichia coli luxAB transcriptional gene fusion library, our laboratory identified two clones whose luminescence increased in the presence of sodium selenite. The clones were designated strains LF20113 and LF20116. Cloning and sequencing portions of the two genes revealed that luxAB had inserted within two previously uncharacterized open reading frames, designated o219 and o393, which we have now named ginS and gutS, respectively. Transcription from these genes in response to increasing concentrations of sodium selenite was confirmed by RNA dot blot analysis. Primer extension analysis of the predicted promoter region of ginS revealed that the promoter was within the previously predicted protein coding sequence and thus, this gene is smaller than originally suspected. Expression of the GinS polypeptide in vivo demonstrated that the protein is approximately 22 kDa, which is also consistent with the smaller coding sequence. Homology searches have also shown that this protein is a member of the DedA family of proteins whose functions remain unknown. We have shown that three genes are down-regulated upon expression of the ginS gene product. These genes are clpX, b1169 , and gspC, and may indicate the involvement of ginS in a new pathway of protein transport in response to selenite exposure. Moreover, strain LF20113 is more sensitive to elevated concentrations of sodium selenite than the parental strain indicating a protective function for the gene. Characterization of the second selenite-responsive gene, gutS, revealed that this gene is not only transcribed in the presence of selenite, but also tellurite, as shown by measurement of luminescence from strain LF20116 as well as by measurement of transcription of gutS by RNA dot blot analysis. Exp

Living organisms must monitor and respond to a diverse array of environmental stimuli. These responses, many of which are genetically-programmed, allow the organisms to obtain nutrients as well as cope with toxins. To identify such responses in Escherichia coli, a chromosomal luxAB gene fusion library was constructed. By screening this library with metals and monitoring for increases in luminescence, two metal-inducible clones were identified. Luminescence of clone LF20110 increases with increasing concentrations of added aluminum, iron, indium, gallium and vanadium, but not added calcium, magnesium or nickel. Luminescence of clone TBT1 increases with increasing concentrations of added tributyltin, triethyltin, tri-n-propyltin and carbonyl cyanide m-chlorophenyl hydrazone, but not added dibutyltin or tetrabutyltin. / Analysis of clone TBT1 revealed a luxAB fusion to the uhpT gene, which encodes a hexose-6-phosphate transport protein. Transcriptional regulation of uhpT by glucose-6-phosphate has been well characterized. However, it had not been previously demonstrated that addition of tributyltin could induce transcription of uhpT. This effect on uhpT is associated with the uncoupling of oxidative phosphorylation. / Isolation and sequencing of the luxAB gene fusion from clone LF20110 uncovered a previously uncharacterized gene, which we call ais (a&barbelow;luminum and i&barbelow;ron s&barbelow;timulated). The ais gene has been sequenced, the transcriptional start site has been identified, and the 22 kDa Ais protein has been overexpressed in vivo. Mutation of the basS gene abrogates metal-induced transcription of ais. However, expression of the BasR-BasS two-component regulatory system, in trans, restores the metal-inducible response. / The E. coli BasR-BasS regulon is poorly characterized. To identify genes regulated by this two-component system, a MudI (lac Ap) gene fusion library was constructed and screened Gene fusions to blc, glmUS, iap, and ygjlJK exhibited increased beta-galactosidase activity upon overexpression of BasR-BasS, while expression of a dsdXA fusion decreased. By searching the E. coli genome database for putative BasR binding sequences, four additional genes, seiC, yibD, yhhT and yaf were identified. Of these nine genes, four have not yet been characterized. The other five are associated with cell membrane functions or sugar metabolism.

Luminescence is a secondary metabolic process that can be detected and assayed in a non-disruptive, in vivo, and real-time manner. As such the Vibrio harveyi luminescence (lux) system constitutes a model system that can be used to delineate the relatively complex transcriptional control mechanisms that are inherent to secondary metabolic processes. Molecular insights into the control of luminescence induction in V. harveyi is limited to the two-component quorum-sensing system and LuxR. While the quorum-sensing system is thought to facilitate luminescence induction through a mechanism of derepression, LuxR has been shown to be an essential activator of the process by virtue of the absence of luminescence in a luxR null mutant. Additionally, it has been suggested that LuxR activates its own expression. Data presented here demonstrates that LuxR is involved in autorepression. Moreover, it is argued that LuxR may not be a typical activator in that it may mediate activation of lux gene expression through a mechanism of repression. An implication of this proposal is that a second unidentified activator is required for luminescence induction to occur. The search of candidates to fill this proposed activation role focused on two highly conserved and extensively studied activators, MetR and CRP. Both are involved in nutrient dependent pathways as MetR is an activator of methionine biosynthesis genes and CRP is implicated in the regulation of metabolic pathways in response to the presence of preferred carbohydrates. In the specific case of the V. harveyi lux system, MetR is shown to act as a repressor, while CRP is shown to be a critical activator of luminescence induction. The evolutionary and ecological implications of these findings are discussed.

The nucleotide sequence from the downstream region of the lux operon from the bioluminescent bacterium Photobacterium phosphoreum was determined. This area contains three genes involved in bioluminescence metabolism: luxF, luxE, and luxG. / LuxF is a new lux gene not found in the extensively studied luminescent bacteria of the Vibrio genus. The 26 kDa LuxF protein was found to be homologous to the 35 kDa $ beta$ subunit of bacterial luciferase (LuxB). LuxF is proposed to have arisen from luxB by a gene duplication event followed by deletion of 100 codons near the amino terminal. The LuxF protein was identified as the abundant non-fluorescent flavoprotein of unknown function found in many Photobacterium species. / LuxE codes for the 42 kDa fatty acyl-protein synthetase subunit of the fatty acid reductase complex which produces fatty aldehydes for the luciferase-catalyzed light-emitting reaction. This enzyme catalyzes the ATP-dependent activation of free fatty acids and covalently transfers acyl groups to other protein subunits via an intramolecular cysteine residue. Site-directed mutagenesis of five conserved cysteine residues has identified the site of fatty acyl transfer of the synthetase as Cys$ sp{364}.$ / LuxG is a new lux gene found in the operons of marine but not terrestrial luminescent bacteria. The 26 kDa LuxG protein was found to be highly homologous to an NAD(P)H:flavin oxidoreductase from Escherichia coli. The primary structure homologies implicate this new lux protein in flavin oxidoreduction biochemistry; a role in the luminescent-specific generation of the FMNH$ sb2$ cosubstrate of luciferase in the light-emitting reaction is likely. / Light emission was restored in dark V. harveyi fatty acid reductase mutants by complementation with V. harveyi and P. phosphoreum genes coding for the corresponding inactive enzyme subunit. The variation in fatty aldehyde- and fatty acid-stimulable in vivo luminescence with cell growth (and lux induction) shows that P. phosphoreum subunits interact poorly and in a protein concentration-dependent manner with V. harveyi fatty acid reductase complexes. (Abstract shortened by UMI.)

Neurological diseases are a common consequence of retroviral infections. The pathogenesis of these diseases however remains undetermined. In an attempt to elucidate the mechanisms involved in certain of these disorders, we have used an experimental approach involving transgenic mice. Transgenic animals provide an important tool in the study of retroviral diseases, since they allow us to circumvent the complex process of retroviral infection. In addition, when retroviral sequences are expressed under the regulation of a CNS-specific promoter, transgenic experiments allow us to evaluate the effects of expressing viral gene products in a given CNS cell population. Specific aspects of the neurological disorders caused by HIV-1, HTLV-1, and Cas-Br-E MuLV were evaluated. Transgenic mice experiments were generated in order to study the pathogenesis of the CNS white matter diseases caused by human retroviruses HIV-1 and by HTLV-1, and to evaluate the function of astroglial cells in mediating the CNS disease associated with Cas-Br-E MuLV infection. On the basis of our experimental results, we propose novel pathogenic mechanisms which may contribute to our understanding of the CNS diseases caused by these retroviruses.

D108 is a temperate, transposable bacteriophage of Escherichia coli, that is closely related to phage Mu. Multiple protein-DNA regulatory complexes span the D108 left-end intergenic region during different stages of phage development. A diverging and asymmetric pattern of transcription is initiated from the opposing Pc and Pe promoters in this region. During lysogeny, the phage genome remains quiescent through the action of c repressor, expressed from the Pc promoter. DNase I footprinting revealed that oligomers of c repressor bind two operators (O1 and O2), one of which (O2) overlaps the early promoter (Pe) such that bound c repressor prevented RNA polymerase binding and transcription of the early operon. During lytic growth, transcription of the early (transposase) operon is controlled by the phage encoded ner repressor in conjunction with the host protein IHF. The purified Ner protein, an 8.6 kDa monomer, bound a large region that overlaps the transcription initiation sites for both the repressor (Pc) and early (Pe) promoters. This operator spans five turns of the DNA helix and is composed of two 11 bp inverted repeats separated by an 8 bp AT-rich spacer. Enzymatic and chemical footprinting studies, in addition to protein cross-linking experiments, indicated that the structural features of Ner-DNA binding represent a novel form of such interactions. The apex of the intrinsically bent ner-operator was localized to the AT-rich spacer. This operator was conformationally altered upon Ner binding. In the absence of IHF, bound Ner prevented RNA polymerase from binding to either Pc or Pe. However, in the presence of IHF, which binds and bends the DNA upstream of Pe, RNA polymerase associated with Pe irrespective of the Ner-DNA complex. The IHF-induced DNA structural change may partially relieve Ner repression of the early promoter, thus ensuring stable transposase gene expression during lytic phage development. Molecular analysis of these protein-DNA interactions has

The partition of surfactants and of a biosurfactant-producing microorganism was studied in polyethylene glycol and dextran aqueous two-phase systems. In the presence of sodium phosphate, surfactants distributed themselves according to their charge. Cationic surfactants preferred the bottom phase, while anionic surfactants were attracted to the top phase. Increasing the phosphate molarity, the pH, the polymer concentration or molecular weight all resulted in a more one-sided surfactant partitioning. Biosurfactant partition was weaker than synthetic surfactant partition due to their weaker effective charge and lack of strong specific affinity for one of the polymers. / Bacillus subtilis cells partitioned very strongly to the bottom phase. Its biosurfactant, surfactin, was found in slightly larger quantities in the top phase. Batch fermentations were carried out in an aqueous two-phase system. Bacterial growth was not inhibited by the high polymer concentration. Surfactin was produced earlier and in larger quantities than in the regular mineral salts medium.

Measles virus is an animal enveloped virus that is a member of the genus morbillivirus in the paramyxoviridae family. Its envelope contains two surface glycoproteins H and F which are required for viral attachment and entry respectively. Virus penetration occurs via a process which involves fusion of the viral membrane with the plasma membrane at the cell surface. Replication of the virus thus follows and leads to giant cell (syncytia) formation. / The infectivity of measles virus is dependent upon a host proteolytic cleavage of the F$ sb0$ glycoprotein into two active subunits F$ sb1$ and F$ sb2$. This cleavage was later shown to expose a hydrophobic sequence at a NH$ sb2$ terminal of the F$ sb1$ which is directly involved in cell fusion and virus penetration. / In order to increase our knowledge concerning cell mediated fusion events we have expressed the fusion glycoprotein of measles virus in insect and human cells by using recombinant baculo- and adenoviruses respectively. Analysis by SDS-PAGE demonstrated that our protein was first synthesized as a 60 Kd protein and cleaved subsequently into its two respective subunits F$ sb1$ and F$ sb2$ of 40 Kd and 20 Kd respectively. Hemolysis assays confirmed the biological activity of this protein in both systems. However, the fusion protein was unable to fuse insect cells.

It has been observed that the class III bacteriophages Mu and D108 and the class II elements, Tn3, Tn1000, IS101 and Tn951, share the characteristics of replicatively transposing to random positions, producing 5 bp target site duplications and of containing the sequence 5$ sp prime$-PUCGAAAPu-3$ sp prime$ at bp 21 from their ends. These shared characteristics led to the hypothesis that these class III and class II elements evolved from a common ancestor transposon. Functional evidence which supports this hypothesis was obtained using the band retardation and in vivo transposition-mating assays. / Mu and D108 transposase proteins were shown to mediate the formation of specific protein-DNA complexes with the ends of Tn3 and IS101, but not with the ends of the IS102 element (class I) demonstrating the conservation of the first step in the transposition reaction between Mu, D108, Tn3 and IS101. / Transposition-mating assays with Mu transposase and the Tn3kan element demonstrated that recombination (rec A independent) products between Tn3kan-containing plasmids and the target (pOX38cam) were formed in the absence of Mu transposase, but their formation was stimulated 200-fold in the presence of Mu transposase protein. These transposition-mating products were resolved in rec $A sp+$ hosts, to generate resolution products suggestive of replicative transposition. Upon subsequent analysis, it was shown that the resolved products (pOX38cam::Tn3kan) had not been cleaved precisely at the Tn3kan element ends, but in the adjacent donor plasmid (pDV-cam) sequences. / Similar experiments with the IS101 element demonstrated that plasmids containing IS101 plus adjacent pSC101 sequences (bp 2233 to 2424), including a formerly unknown, but functional (bound by IHF) IHF site (bp 2238-2251), produced resolvable transposition-mating products in the presence of Mu transposase. These resolution products suggested that IS101 (like Tn3kan) was replicatively transposed. / The results presented provide functional evidence that links (evolutionarily) the class III transposable phages Mu and D108 and the class II elements Tn3 and IS101.