Biomedical subjects

S B Jovanovich

Publications and source records attributed to S B Jovanovich.

13 recordsLinked to original sources

PubMed2001

Identification of toxicologically predictive gene sets using cDNA microarrays.

We have developed an approach to classify toxicants based upon their influence on profiles of mRNA transcripts. Changes in liver gene expression were examined after exposure of mice to 24 model treatments that fall into five well-studied toxicological categories: peroxisome proliferators, aryl hydrocarbon receptor agonists, noncoplanar polychlorinated biphenyls, inflammatory agents, and hypoxia-inducing agents. Analysis of 1200 transcripts using both a correlation-based approach and a probabilistic approach resulted in a classification accuracy of between 50 and 70%. However, with the use of a forward parameter selection scheme, a diagnostic set of 12 transcripts was identified that provided an estimated 100% predictive accuracy based on leave-one-out cross-validation. Expansion of this approach to additional chemicals of regulatory concern could serve as an important screening step in a new era of toxicological testing.

Animals↗

PubMed2000

Sub-microliter DNA sequencing for capillary array electrophoresis.

DNA sequencing from sub-microliter samples was demonstrated for capillary array electrophoresis by optimizing the analysis of 500 nl reaction aliquots of full-volume reactions and by preparing 500 nl reactions within fused-silica capillaries. Sub-microliter aliquots were removed from the pooled reaction products of 10 microl dye-primer cycle-sequencing reactions and analyzed without modifying either the reagent concentrations or instrument workflow. The impact of precipitation methods, resuspension buffers, and injection times on electrokinetic injection efficiency for 500 nl aliquots were determined by peak heights, signal-to-noise ratios, and changes in base-called readlengths. For 500 nl aliquots diluted to 5 microl in 60% formamide-1 mM EDTA and directly injected, a five-fold increase in signal-to-noise ratios was obtained by increasing injection times from 10 to 80 s without a corresponding increase in peak widths or reduction in readlengths. For 500 nl aliquots precipitated in alcohol, 80 +/- 5% template recovery and a two-fold decrease in conductivity was obtained, resulting in a two-fold increase in peak heights and 50 to 100 bases increase in readlengths. In a comparison of aliquot volumes and precipitation methods, equivalent readlengths were obtained for 500 nl, 4 microl, and 8 microl aliquots by simply adjusting the electrokinetic injection conditions. To ascertain the robustness of this methodology for genomic sequencing, 96 Arabidopsis thaliana subclones were sequenced, with a yield of 38 624 bases obtained from 500 nl aliquots versus 30 764 bases from standard scale reactions. To demonstrate 500 nl sample preparation, reactions were performed in fused-silica capillary reaction chambers using air-based thermal cycling. A readlength of 690 bases was obtained for the polymerase chain reaction product of an Arabidopsis subclone without modifying the reagent concentrations, post-reaction processing or electrokinetic injection workflow. These results demonstrated the fundamental feasibility of small-volume DNA sequencing for high-throughput capillary electrophoresis.

Arabidopsis↗

PubMed2000

Bacterial rhodopsin: evidence for a new type of phototrophy in the sea.

Extremely halophilic archaea contain retinal-binding integral membrane proteins called bacteriorhodopsins that function as light-driven proton pumps. So far, bacteriorhodopsins capable of generating a chemiosmotic membrane potential in response to light have been demonstrated only in halophilic archaea. We describe here a type of rhodopsin derived from bacteria that was discovered through genomic analyses of naturally occuring marine bacterioplankton. The bacterial rhodopsin was encoded in the genome of an uncultivated gamma-proteobacterium and shared highest amino acid sequence similarity with archaeal rhodopsins. The protein was functionally expressed in Escherichia coli and bound retinal to form an active, light-driven proton pump. The new rhodopsin exhibited a photochemical reaction cycle with intermediates and kinetics characteristic of archaeal proton-pumping rhodopsins. Our results demonstrate that archaeal-like rhodopsins are broadly distributed among different taxa, including members of the domain Bacteria. Our data also indicate that a previously unsuspected mode of bacterially mediated light-driven energy generation may commonly occur in oceanic surface waters worldwide.

Aerobiosis↗

PubMed2000

Automated parallel DNA sequencing on multiple channel microchips.

We report automated DNA sequencing in 16-channel microchips. A microchip prefilled with sieving matrix is aligned on a heating plate affixed to a movable platform. Samples are loaded into sample reservoirs by using an eight-tip pipetting device, and the chip is docked with an array of electrodes in the focal plane of a four-color scanning detection system. Under computer control, high voltage is applied to the appropriate reservoirs in a programmed sequence that injects and separates the DNA samples. An integrated four-color confocal fluorescent detector automatically scans all 16 channels. The system routinely yields more than 450 bases in 15 min in all 16 channels. In the best case using an automated base-calling program, 543 bases have been called at an accuracy of >99%. Separations, including automated chip loading and sample injection, normally are completed in less than 18 min. The advantages of DNA sequencing on capillary electrophoresis chips include uniform signal intensity and tolerance of high DNA template concentration. To understand the fundamentals of these unique features we developed a theoretical treatment of cross-channel chip injection that we call the differential concentration effect. We present experimental evidence consistent with the predictions of the theory.

Automation↗

PubMed2000

Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage.

Cultivation-independent surveys of ribosomal RNA genes have revealed the existence of novel microbial lineages, many with no known cultivated representatives. Ribosomal RNA-based analyses, however, often do not provide significant information beyond phylogenetic affiliation. Analysis of large genome fragments recovered directly from microbial communities represents one promising approach for characterizing uncultivated microbial species better. To assess further the utility of this approach, we constructed large-insert bacterial artificial chromosome (BAC) libraries from the genomic DNA of planktonic marine microbial assemblages. The BAC libraries we prepared had average insert sizes of 80 kb, with maximal insert sizes > 150 kb. A rapid screening method assessing the phylogenetic diversity and representation in the library was developed and applied. In general, representation in the libraries agreed well with previous culture-independent surveys based on polymerase chain reaction (PCR)amplified rRNA fragments. A significant fraction of the genome fragments in the BAC libraries originated from as yet uncultivated microbial species, thought to be abundant and widely distributed in the marine environment. One entire BAC insert, derived from an uncultivated, surface-dwelling euryarchaeote, was sequenced completely. The planktonic euryarchaeal genome fragment contained some typical archaeal genes, as well as unique open reading frames (ORFs) suggesting novel function. In total, our results verify the utility of BAC libraries for providing access to the genomes of as yet uncultivated microbial species. Further analysis of these BAC libraries has the potential to provide significant insight into the genomic potential and ecological roles of many indigenous microbial species, cultivated or not.

Archaea↗

PubMed1990

Identification of a heat shock promoter in the topA gene of Escherichia coli.

The transcriptional activity of the topA gene which codes for topoisomerase I was examined. An in vitro assay determined that the P1 promoter was dependent on the sigma 32 subunit of RNA polymerase. The transcriptional activity of the four topA promoters was examined by nuclease S1 mapping of the transcripts during a heat shock. This sigma 32-dependent promoter was shown to function as a heat shock promoter, although topoisomerase I is not a heat shock protein. A possible method of compensation of transcription activity by the other promoters to maintain the level of topoisomerase I during heat shock is proposed.

Base Sequence↗

PubMed1990

Cloning and in vivo and in vitro regulation of cyclic AMP-dependent carbon starvation genes from Escherichia coli.

The regulation of three Escherichia coli carbon starvation (cst) genes fused to lacZ was examined. Expression of these genes is induced by starvation for a carbon source. The role of carbon and cyclic AMP (cAMP) availability and of an altered-function crp mutation were investigated for their effect on cst expression in vivo. The experiments indicated that cAMP concentrations controlled the absolute expression of one cst fusion, but the other two cst fusions were dependent upon some component not present in exponentially growing cells under conditions of glucose excess, even when cAMP was added. To examine the regulation of these genes in further detail, the three cst::lacZ fusions were cloned on multicopy plasmids. All three cst::lacZ fusions retained their inducible regulatory phenotype in the multicopy state. Analysis of the expression of the cloned cst::lacZ fusions in an in vitro-coupled transcription-translation cell-free system demonstrated that the predominant promoter(s) present on each cloned DNA was dependent on sigma 70 for expression. In vitro cAMP titration curves indicated that this molecule was necessary and sufficient for the expression of one fusion but not sufficient for the second fusion, while the third fusion exhibited constitutive levels of expression in vitro. The results are discussed in the context of the E. coli carbon starvation response.

Antibodies, Monoclonal↗

PubMed1989

Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters.

Four pseudorevertants of a -10 region lacP mutation were isolated. Three of these mutations were found to activate nascent promoters. These mutations were: a -2 G/C----A/T change (-2A) promoting transcription at position +11, a +1 A/T----T/A change (+1T) promoting transcription initiation at position +13, and a +10 C/G----A/T change (+10A) promoting transcription initiation at a complex series of positions. The fourth mutation [a -12 T/A----A/T change (-12A)] promotes transcription initiation at -1. The promoters activated by mutations -12A, -2A and +1T resembled the canonical sigma 70 promoter sequences. The +10A promoter activity is also dependent upon the sigma 70 holoenzyme but can not be readily assigned to a specific promoter sequence.

Escherichia coli↗

PubMed1989

In an Escherichia coli coupled transcription-translation system, expression of the osmoregulated gene proU is stimulated at elevated potassium concentrations and by an extract from cells grown at high osmolality.

We have studied the in vitro expression of the osmoregulated proU promoter in Escherichia coli coupled transcription-translation (S-30) extracts as a function of the osmolality of the culture medium used to grow the cells and of the salt concentration added to the extract. These variables represent novel extensions of the use of S-30 extracts to investigate gene regulatory phenomena in vitro. The concentrations of potassium acetate and of the physiologically relevant osmolyte potassium glutamate for maximal expression of the proU promoter are approximately 2-fold higher than the concentrations of these salts providing maximal expression of the lacUV5 promoter. The relative promoter activity of proU with respect to lacUV5 increases more than 30-fold with an increase in salt concentration from 50 to 300 mM. In comparative studies with S-30 extracts prepared from cells grown at high and low osmolalities, we find that at fixed salt concentrations expression of proU is increased 10-fold in the S-30 extract prepared from high osmolality-grown cells, whereas the expression of lacUV5 is increased less than 2-fold. Addition of anti-sigma 70 monoclonal antibodies or purified sigma 70 to the S-30 extract demonstrated that the major proU promoter(s) used in the S-30 extracts is sigma 70-dependent.

Acetates↗

PubMed1989

In vitro use of monoclonal antibodies in Escherichia coli S-30 extracts to determine the RNA polymerase sigma subunit required by a promoter.

RNA polymerase requires one of a family of sigma subunits for specific promoter recognition and initiation. We have developed an in vitro method to define the RNA polymerase sigma subunit required by a promoter. Mouse monoclonal antibodies specific for either Escherichia coli sigma 70 or sigma 32 or Salmonella typhimurium sigma 54 were added to an E. coli coupled transcription-translation S-30 extract programed with a DNA template containing the promoter of interest. Using the representative lacUV5, glnAP2, and rpoDHS promoters as controls, we found that monoclonal antibodies to a given sigma subunit strongly inhibited transcription from cognate promoters which utilized that sigma subunit, but had little effect on transcription from noncognate promoters which used other sigma subunits. Supplementation of the S-30 extract with purified sigma 70, sigma 54, or RNA polymerase sigma 32-holoenzyme stimulated expression from the cognate promoters and inhibited noncognate promoters. These two tests, addition of monoclonal antibodies and addition of sigma subunits, provide a rapid means of identifying whether the sigma subunit required by any promoter expressed in the S-30 extract is sigma 70, sigma 54, or sigma 32. We suggest that this method may provide a systematic approach for identifying promoters which use as yet uncharacterized sigma subunits.

Antibodies, Monoclonal↗

PubMed1988

Rapid response to osmotic upshift by osmoregulated genes in Escherichia coli and Salmonella typhimurium.

The rapid in vivo response of both Escherichia coli and Salmonella typhimurium osmoregulated genes to an osmotic upshift was analyzed in detail by using chromosomal operon fusions. Within 10 min after the addition of 0.3 M NaCl to the culture medium, the differential rates of expression of both an S. typhimurium proU-lac fusion and a proP-lac fusion increased by 180- and 17-fold respectively, while an E. coli ompC-lac fusion increased by 3.4-fold. For all three stimulated promoters, the increased rate of expression was maintained until about 40 min after the osmotic upshift. Thereafter, proU expression continued at a steady-state rate that was 27-fold higher than that of the control, while proP and ompC expression fell to 1.4- and 2-fold of the control rates, respectively. In contrast, expression of an E. coli ompF-lac fusion decreased twofold within 2.5 min. For proU, the length of the lag phase, which preceded the onset of the rapid response, increased with the degree of osmotic upshift, above a threshold of 0.2 M NaCl; the onset of the rapid proU response also preceded the resumption of growth. The rapid response phase, which was first quantitated for proU, proP, ompC, and ompF in this study, is an important component of the osmoregulation of these promoters. The addition of the osmoprotectant glycine betaine at the time of osmotic upshift decreased both the length of the rapid response and the subsequent steady-state of expression of proU.

Bacterial Outer Membrane Proteins↗

PubMed1987

Estimation of the effect of coumermycin A1 on Salmonella typhimurium promoters by using random operon fusions.

We have estimated the extent to which relaxation of supercoiling by the DNA gyrase inhibitor coumermycin A1 affects gene expression in vivo in Salmonella typhimurium. We isolated a set of Mu d1-8 Lac+ operon fusions to random promoters and measured the effect of coumermycin A1 on the expression of 67 fusions. The differential rate of synthesis was increased for 70% of the fusions and decreased for 16%, and 13% of the fusions had less than a 25% change in expression. The coumermycin A1 response was found to correlate well (P = 0.067) with the basal level of expression such that coumermycin A1 tended to stimulate fusions with low expression and inhibit those with high expression. Since the vast majority of the fusions were sensitive to coumermycin A1 addition and, therefore, to the level of supercoiling, these results indicate that if the level of supercoiling were to vary under physiological conditions, then major readjustments in the cellular economy would occur.

Aminocoumarins↗

PubMed1985

Regulation of a cya-lac fusion by cyclic AMP in Salmonella typhimurium.

cya-lac and crp-lac operon fusions were isolated in Salmonella typhimurium by using the phage Mu d1(lac cts Apr). Both transduction and reversion analyses have indicated that lac expression is controlled by the appropriate promoter, e.g., either crpp or cyap. By using chromosomal mobilization techniques, we found that cya had a clockwise direction of transcription on the standard S. typhimurium map. The cya-lac fusions could be complemented by Escherichia coli F'133, which covers cya, with a resultant 17 to 38% decrease in cya expression. Cyclic AMP was found to be able to repress the expression of the cya-lac fusion ninefold when present at 25 mM. This repression was not seen in crp backgrounds, and hence is mediated by the cAMP receptor protein. Repression of cya was also found upon growth on carbon sources known to elicit high cyclic AMP levels.

Adenylyl Cyclases↗