Regulation of the expression of {\it mutS, mutL}, and {\it mutH\/} mismatch repair genes in {\it Escherichia coli\/} K-12

Post-replication DNA mismatch repair plays crucial roles in mutation avoidance and maintenance of chromosome stability in both prokaryotes and eukaryotes. In humans, deficiency in this repair system leads to a predisposition for certain cancers. The biochemistry of this repair system has been best studied in a model bacterium Escherichia coli. In this thesis, regulation of expression of mutS, mutL and mutH genes, whose products mediate methyl-directed mismatch (MDM) repair in E. coli, is investigated. One-step affinity purification schemes were developed to purify E. coli MutS, MutL and MutH proteins fused to a His-6-affinity tag. His-6-MutS exhibited the same mismatch binding activity and specificity as the native MutS protein. Purified His-6-MutS, -MutL and -MutH proteins were used to develop quantitative Western blotting assays for amounts of MutS, MuL and MutH proteins under various conditions. It was found that the three proteins were present in relatively low amounts in exponentially growing cells and MutS and MutH were diminished in stationary-phase cells. Further studies indicated that the drop in the amounts of MutS and MutH proteins in stationary-phase cells was mediated through RpoS, a key global regulator of stationary-phase transition. In both exponential- and stationary-phase cells, MutS amount was also negatively regulated by the Hfq (HF-I) global regulator, which is required for RpoS translation, through an RpoS-independent mechanism. $\beta$-galactosidase assays of mutS-lacZ operon and gene fusions suggested that hfq regulates mutS posttranscriptionally, and RNase T2 protection assays revealed that Hfq destabilizes mutS transcripts in exponentially growing cells. To study the relation between regulation of MDM repair and mutagenesis, amounts of MutS, MutL and MutH were measured in starved cells undergoing adaptive mutagenesis. It was found that MutS amount dropped drastically, MutH amount dropped slightly, whereas MutL amount remained essentially constant in starved cells. Overexpression of MutL did not reverse the drop in the amounts of MutS or MutH protein. These results ruled out several explanations for a phenomenon in which overexpression of MutL, but not MutS, reversed adaptive mutagenesis. The findings further suggested that functional MutL is limiting during adaptive mutagenesis. The implications of regulation of the MDM repair are discussed in the context of mutagenesis, pathogenesis and tumorigenesis. ^

Regulation of pyridoxal $5\prime$-phosphate biosynthesis in {\it Escherichia coli\/} K-12

Vitamin B$\sb6$ (or pyridoxal 5$\sp\prime$-phosphate, PLP) is an essential, ubiquitous coenzyme that affects many aspects of amino acid and cellular metabolism in all organisms. The goal of this thesis is to examine the regulation of PLP biosynthesis in Escherichia coli K-12. First, PdxH oxidase is a PLP biosynthetic enzyme, which uses molecular oxygen as an electron acceptor under aerobic assay conditions. To test if facultative anaerobic E. coli uses another enzyme to replace the function of PdxH oxidase anaerobically, suppressors of a pdxH null mutant were isolated anaerobically after 2-aminopurine or spontaneous mutagenesis. Only one specific bypass mutation in another PLP biosynthetic gene pdxJ was found, suggesting that PdxH oxidase is able to function anaerobically and PdxT utilizes D-1-deoxyxyulose as a substrate. Second, regulation of the serC (pdxF)-aroA operon, which is involved the biosynthesis of L-serine, PLP and aromatic compounds was examined. A serC (pdxF) single gene transcript and a serC (pdXf)-aroA cotranscript initiated at P$\sb{serC\ (pdxF)}$ upstream of serC (pdxF) were detected. The expression of the operon is activated by leucine responsive regulatory protein (LRP) and repressed by cAMP receptor protein-cAMP complex (CRP$\cdot$cAMP) at the transcriptional level. LRP activates the operon by directly binding to the upstream consensus box. Binding of CRP$\cdot$cAMP to the upstream CRP box diminishes the activation effect of LRP. However, deletion of the CRP box did not affect the repression of CRP$\cdot$cAMP, suggesting that CRP$\cdot$cAMP may repress the operon indirectly by stimulating the activity or level of an unidentified repressor. The overall effect of this regulation is to maximize the expression of the operon when the cells are growing in minimal-glucose medium. In addition, the binding and the transcription of P$\sb{serC\ (pdxF)}$ by RNA polymerase require a supercoiled circular DNA, indicating that DNA supercoiling affects the transcription of the operon. Third, regulation of another PLP biosynthetic gene gapB was also examined. gapB is activated by CRP$\cdot$cAMP and repressed by catabolic repressor activator protein (CRA). However, the activation of CRP$\cdot$cAMP is epistatic to the repression of CRA. Due to the CRA repression, gapB was expressed at a low level in all the media tested, suggesting that it may be the rate-limiting step of PLP biosynthesis. In summary, unlike genes in many biosynthetic pathways, PLP biosynthetic genes are regulated by global regulators that are important for carbon and amino acid metabolism, instead of the end product(s) of the pathway. ^

Cloning, sequencing and partial functional characterization of the 5' region of the human p75 tumor necrosis factor receptor-encoding gene (TNF-R)

A 1887-bp region at the 5' flank of the human p75 tumor necrosis factor receptor (p75 TNF-R)-encoding gene was found to be active in driving expression of the luc (luciferase-encoding) reporter gene, suggesting that it contains the promoter for the receptor. Rather unexpectedly, a 1827-bp region at the 3' end of the first intron of the p75 TNF-R gene also displayed promoter activity. This activity may be artefactual, reflecting only the presence of an enhancer in this region; yet it also raises the possibility that p75 TNF-R is controlled by more than one promoter and that it encodes various forms of the receptor, or even other proteins. We present here the nucleotide sequences of the 5' flanking and intron regions. Possible implications for the transcriptional regulation of the p75 TNF-R gene are discussed.

Using Informatics and the Electronic Medical Record to Describe Antimicrobial Use in the Clinical Management of Diarrhea Cases at 12 Companion Animal Practices

Antimicrobial drugs may be used to treat diarrheal illness in companion animals. It is important to monitor antimicrobial use to better understand trends and patterns in antimicrobial resistance. There is no monitoring of antimicrobial use in companion animals in Canada. To explore how the use of electronic medical records could contribute to the ongoing, systematic collection of antimicrobial use data in companion animals, anonymized electronic medical records were extracted from 12 participating companion animal practices and warehoused at the University of Calgary. We used the pre-diagnostic, clinical features of diarrhea as the case definition in this study. Using text-mining technologies, cases of diarrhea were described by each of the following variables: diagnostic laboratory tests performed, the etiological diagnosis and antimicrobial therapies. The ability of the text miner to accurately describe the cases for each of the variables was evaluated. It could not reliably classify cases in terms of diagnostic tests or etiological diagnosis; a manual review of a random sample of 500 diarrhea cases determined that 88/500 (17.6%) of the target cases underwent diagnostic testing of which 36/88 (40.9%) had an etiological diagnosis. Text mining, compared to a human reviewer, could accurately identify cases that had been treated with antimicrobials with high sensitivity (92%, 95% confidence interval, 88.1%-95.4%) and specificity (85%, 95% confidence interval, 80.2%-89.1%). Overall, 7400/15,928 (46.5%) of pets presenting with diarrhea were treated with antimicrobials. Some temporal trends and patterns of the antimicrobial use are described. The results from this study suggest that informatics and the electronic medical records could be useful for monitoring trends in antimicrobial use.

2-methylthio-N$\sp6$-dimethylallyl modification of adenosine 37 of tRNAs in {\it Escherichia coli\/} K-12

The hypermodified, hydrophobic 2-methylthio-N$\sp6$-(dimethylallyl)-adenosine (ms${2{\cdot}6}\atop1$A) residue occurs $3\sp\prime$ to the anticodon in tRNA species that read codons beginning with U. The first step (i$\sp6$A37 formation) of this modification is catalyzed by dimethylallyl diphosphate:tRNA dimethyallyltransferase (EC 2.5.1.8), which is the product of the miaA gene. Subsequent steps were proposed to be catalyzed by MiaB and MiaC enzymes to complete the ms${2{\cdot}6}\atop1$A37 modification. The study of functions of the ms${2{\cdot}6}\atop1$A37 is very important because this modified base is one of the best candidates for a role in global control in response to environmental stress. This dissertation describes the further delineation of functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli K-12 cells. This work provides significant information on functions of tRNA modifications in E. coli cells to adapt to stressful environmental conditions. Three hypotheses were tested in this work.^ The first hypothesis tested was that non-optimal translation processes cause increased spontaneous mutagenesis by the induction of SOS response in starving cells. To test this hypothesis, I measured spontaneous mutation rates of wild type cells and various mutant strains which are defective in tRNA modification, SOS response, or oxidative damage repair. I found that the miaA mutation acts as a mutator that increased Lac$\sp+$ reversion rates and Trp$\sp+$ reversion frequencies of the wild-type cells in starving conditions. However, the lexA3(Ind)(which abolishes the induction of SOS response) mutation abolished the mutator phenotype of the miaA mutant. The recA430 mutation, not other identified SOS genes, decreased the Lac$\sp+$ reversion to a less extent than that of the lexA3(Ind) mutation. These results suggest that RecA together with another unidentified SOS gene product are responsible for the process.^ The second hypothesis tested was that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ molecules in form of a protein dimer. To test this hypothesis, three versions of the MiaA protein and seven species of tRNA substrates were purified. Binding studies by gel mobility shift assays, filter binding assays and gel filtration shift assays support the hypothesis that MiaA protein binds to full-length tRNA$\sp{\rm Phe}$ as a protein dimer but as a monomer to the anticodon stem-and-loop. These results were further supported by using steady state enzyme kinetic studies.^ The third hypothesis tested in this work was that the miaB gene in E. coli exists and is clonable. The miaB::Tn10dCm insertion mutation of Salmonella typhimurium was transduced to E. coli K-12 cells by using P$\sb1$ and P$\sb{22}$ bacteriophages. The insertion was confirmed by HPLC analyses of nucleotide profiles of miaB mutants of E. coli. The insertion mutation was cloned and DNA sequences adjacent to the transposon were sequenced. These DNA sequences were 86% identical to the f474 gene at 14.97 min chromosome of E. coli. The f474 gene was then cloned by PCR from the wild-type chromosome of E. coli. The recombinant plasmid complemented the mutant phenotype of the miaB mutant of E. coli. These results support the hypothesis that the miaB gene of E. coli exists and is clonable. In summary, functions of the ms${2{\cdot}6}\atop1$A37 modification in E. coli cells are further delineated in this work in perspectives of adaptation to stressful environmental conditions and protein:tRNA interaction. (Abstract shortened by UMI.) ^

The distribution of oxygen and nutrients in the Persian Gulf according to observations of R.V. "Meteor" in spring 1965

The Persian Gulf situated in the arid climate region of the northern hemisphere shows special conditions in its hydrochemistry. The high evaporation, the lack of large rivers, and the exclusion of deep water from the Indian Ocean governs the nutrient cycle. At 28 stations in the deeper part of the Persian Gulf (Iran side), in the Strait of Hormuz, and in the Gulf of Oman determinations of dissolved oxygen, dissolved inorganic phosphate, silicate, and pH were carried out. On 4 selected transverse profiles for phosphate, and dissolved oxygen and on 1 length profile for phosphate, silicate, oxygen, and pH the distribution of these components is shown and the in- and outflow is characterized. It is also pointed out that the nutrients on their way into the Persian Gulf are diminished and that temporary replenishment supply from a layer of about 100 m depth in the Indian Ocean follows. On one horizontal map the phosphate distribution in the surface and 30 m layer gives reference to biological activity. One diagram where nitrogen components are plotted against phosphate shows that nitrate is a limiting factor for productivity. O2/PO4-P and PO4-P/S? diagrams enable the different waterbodies and mixed layers to be characterized.

(Table 1) Sea ice and snow characteristics and heat fluxes observed during R/V Aurora Australis cruise to East Antarctica in September/October 2007

The properties of snow on East Antarctic sea ice off Wilkes Land were examined during the Sea Ice Physics and Ecosystem Experiment (SIPEX) in late winter of 2007, focusing on the interaction with sea ice. This observation includes 11 transect lines for the measurement of ice thickness, freeboard, and snow depth, 50 snow pits on 13 ice floes, and diurnal variation of surface heat flux on three ice floes. The detailed profiling of topography along the transects and the d18O, salinity, and density datasets of snow made it possible to examine the snow-sea-ice interaction quantitatively for the first time in this area. In general, the snow displayed significant heterogeneity in types, thickness (mean: 0.14 +- 0.13 m), and density (325 +- 38 kg/m**3), as reported in other East Antarctic regions. High salinity was confined to the lowest 0.1 m. Salinity and d18O data within this layer revealed that saline water originated from the surface brine of sea ice in 20% of the total sites and from seawater in 80%. From the vertical profiles of snow density, bulk thermal conductivity of snow was estimated as 0.15 W/K/m on average, only half of the value used for numerical sea-ice models. Although the upward heat flux within snow estimated with this value was significantly lower than that within ice, it turned out that a higher value of thermal conductivity (0.3 to 0.4 W/K/m) is preferable for estimating ice growth amount in current numerical models. Diurnal measurements showed that upward conductive heat flux within the snow and net long-wave radiation at the surface seem to play important roles in the formation of snow ice from slush. The detailed surface topography allowed us to compare the air-ice drag coefficients of ice and snow surfaces under neutral conditions, and to examine the possibility of the retrieval of ice thickness distribution from satellite remote sensing. It was found that overall snow cover works to enhance the surface roughness of sea ice rather than moderate it, and increases the drag coefficient by about 10%. As for thickness retrieval, mean ice thickness had a higher correlation with ice surface roughness than mean freeboard or surface elevation, which indicates the potential usefulness of satellite L-band SAR in estimating the ice thickness distribution in the seasonal sea-ice zone.

Description and illustrations of Mn deposits from the John Murray Expedition (R/V Mabahiss)

During the cruise of the" Mabahiss" from Zanzibar to Colombo at Station 133 (1° 25' 54" S. to 1° 19' 42" S. and 66° 34' 12" E. to 66° 35' 18" E.) several small rock fragments were brought up in the Monegasque net; and, since at this position there is no possibility of the material being transferred by floating Ice, these specimens are of some interest as samples of oceanic rock foundations. All the rocks have a black appearance, but in the majority this skin is of negligible thickness. Exceptionally, however, it may attain to 1/3 in. (St. 133, 8), and then the specimens are rounded. The coating is made of dark opaque manganese material. At Station 166 one or two similar specimens of angular basalt were found in the trawl consisting mainly of manganese nodules.

Early Cenozoic Atlantic and Pacific benthic foraminiferal isotopes from 12 sites

Oxygen and carbon isotope records are important tools used to reconstruct past ocean and climate conditions, with those of benthic foraminifera providing information on the deep oceans. Reconstructions are complicated by interspecies isotopic offsets that result from microhabitat preferences (carbonate precipitation in isotopically distinct environments) and vital effects (species-specific metabolic variation in isotopic fractionation). We provide correction factors for early Cenozoic benthic foraminifera commonly used for isotopic measurements (Cibicidoides spp., Nuttallides truempyi, Oridorsalis spp., Stensioina beccariiformis, Hanzawaia ammophila, and Bulimina spp.), showing that most yield reliable isotopic proxies of environmental change. The statistical methods and larger data sets used in this study provide more robust correction factors than do previous studies. Interspecies isotopic offsets appear to have changed through the Cenozoic, either (1) as a result of evolutionary changes or (2) as an artifact of different statistical methods and data set sizes used to determine the offsets in different studies. Regardless of the reason, the assumption that isotopic offsets have remained constant through the Cenozoic has introduced an 1-2°C uncertainty into deep sea paleotemperature calculations. In addition, we compare multiple species isotopic data from a western North Atlantic section that includes the Paleocene-Eocene thermal maximum to determine the most reliable isotopic indicator for this event. We propose that Oridorsalis spp. was the most reliable deepwater isotopic recorder at this location because it was best able to withstand the harsh water conditions that existed at this time; it may be the best recorder at other locations and for other extreme events also.