Comparison of the 5′ nuclease activities of Taq DNA polymerase and its isolated nuclease domain

Many eubacterial DNA polymerases are bifunctional molecules having both polymerization (P) and 5′ nuclease (N) activities, which are contained in separable domains. We previously showed that the DNA polymerase I of Thermus aquaticus (TaqNP) endonucleolytically cleaves DNA substrates, releasing unpaired 5′ arms of bifurcated duplexes. Here, we compare the substrate specificities of TaqNP and the isolated 5′ nuclease domain of this enzyme, TaqN. Both enzymes are significantly activated by primer oligonucleotides that are hybridized to the 3′ arm of the bifurcation; optimal stimulation requires overlap of the 3′ terminal nucleotide of the primer with the terminal base pair of the duplex, but the terminal nucleotide need not hybridize to the complementary strand in the substrate. In the presence of Mn2+ ions, TaqN can cleave both RNA and circular DNA at structural bifurcations. Certain anti-TaqNP mAbs block cleavage by one or both enzymes, whereas others can stimulate cleavage of nonoptimal substrates.

A genomic approach to gene fusion technology

Gene expression profiling provides powerful analyses of transcriptional responses to cellular perturbation. In contrast to DNA array-based methods, reporter gene technology has been underused for this application. Here we describe a genomewide, genome-registered collection of Escherichia coli bioluminescent reporter gene fusions. DNA sequences from plasmid-borne, random fusions of E. coli chromosomal DNA to a Photorhabdus luminescens luxCDABE reporter allowed precise mapping of each fusion. The utility of this collection covering about 30% of the transcriptional units was tested by analyzing individual fusions representative of heat shock, SOS, OxyR, SoxRS, and cya/crp stress-responsive regulons. Each fusion strain responded as anticipated to environmental conditions known to activate the corresponding regulatory circuit. Thus, the collection mirrors E. coli's transcriptional wiring diagram. This genomewide collection of gene fusions provides an independent test of results from other gene expression analyses. Accordingly, a DNA microarray-based analysis of mitomycin C-treated E. coli indicated elevated expression of expected and unanticipated genes. Selected luxCDABE fusions corresponding to these up-regulated genes were used to confirm or contradict the DNA microarray results. The power of partnering gene fusion and DNA microarray technology to discover promoters and define operons was demonstrated when data from both suggested that a cluster of 20 genes encoding production of type I extracellular polysaccharide in E. coli form a single operon.

NO chemical events in the human airway during the immediate and late antigen-induced asthmatic response

A wealth of evidence supports increased NO (NO⋅) in asthma, but its roles are unknown. To investigate how NO participates in inflammatory airway events in asthma, we measured NO⋅ and NO⋅ chemical reaction products [nitrite, nitrate, S-nitrosothiols (SNO), and nitrotyrosine] before, immediately and 48 h after bronchoscopic antigen (Ag) challenge of the peripheral airways in atopic asthmatic individuals and nonatopic healthy controls. Strikingly, NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{3}^{-}}}\end{equation*}\end{document} was the only NO⋅ derivative to increase during the immediate Ag-induced asthmatic response and continued to increase over 2-fold at 48 h after Ag challenge in contrast to controls [P < 0.05]. NO\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{_{2}^{-}}}\end{equation*}\end{document} was not affected by Ag challenge at 10 min or 48 h after Ag challenge. Although SNO was not detectable in asthmatic airways at baseline or immediately after Ag, SNO increased during the late response to levels found in healthy controls. A model of NO⋅ dynamics derived from the current findings predicts that NO⋅ may have harmful effects through formation of peroxynitrite, but also subserves an antioxidant role by consuming reactive oxygen species during the immediate asthmatic response, whereas nitrosylation during the late asthmatic response generates SNO, safe reservoirs for removal of toxic NO⋅ derivatives.

Stationary-phase mutation in the bacterial chromosome: Recombination protein and DNA polymerase IV dependence

Several microbial systems have been shown to yield advantageous mutations in slowly growing or nongrowing cultures. In one assay system, the stationary-phase mutation mechanism differs from growth-dependent mutation, demonstrating that the two are different processes. This system assays reversion of a lac frameshift allele on an F′ plasmid in Escherichia coli. The stationary-phase mutation mechanism at lac requires recombination proteins of the RecBCD double-strand-break repair system and the inducible error-prone DNA polymerase IV, and the mutations are mostly −1 deletions in small mononucleotide repeats. This mutation mechanism is proposed to occur by DNA polymerase errors made during replication primed by recombinational double-strand-break repair. It has been suggested that this mechanism is confined to the F plasmid. However, the cells that acquire the adaptive mutations show hypermutation of unrelated chromosomal genes, suggesting that chromosomal sites also might experience recombination protein-dependent stationary-phase mutation. Here we test directly whether the stationary-phase mutations in the bacterial chromosome also occur via a recombination protein- and pol IV-dependent mechanism. We describe an assay for chromosomal mutation in cells carrying the F′ lac. We show that the chromosomal mutation is recombination protein- and pol IV-dependent and also is associated with general hypermutation. The data indicate that, at least in these male cells, recombination protein-dependent stationary-phase mutation is a mechanism of general inducible genetic change capable of affecting genes in the bacterial chromosome.

Effects of trace concentrations of anesthetic gases on behavioral performance of operating room personnel /

Mode of access: Internet.

Brief narrative of events touching various reforms /

Last page blank.

Hugh Worthington. A novel.

Mode of access: Internet.

Marian Grey ...

Mode of access: Internet.

An arithmetic unit for on-line computation /

Vita.

A four function calculator /

"Report No. UIUCDCS-R-75-692"

Historical geography of Britain and the British Empire ...

bk.I. The making of England; the making of empire; the establishment of empire, B.C., 55 to A.D. 1815.--bk.II. The expansion and consolidation of the British Empire, A.D. 1800 to present day.

Madeline : a novel /

Mode of access: Internet.

History of the Church of England : from the abolition of the Roman jurisdiction /

Imprint varies: v.3-4, London, New York, H. Frowde ; Oxford, University Press.--v.5-6, Oxford, Clarendon Press.

Hugh Worthington : a novel /

Also published under the titles: Bad Hugh, and Cousin Hugh.