Bacteria employ survival strategies to protect themselves against foreign invaders, including bacteriophages. The ‘immune system’ of bacteria relies mostly on restriction-modification (R-M) systems. The primary role of R-M systems is to protect the host from invading foreign DNA molecules. Three major types of R–M system are found in bacteria, viz.Types I, II and III. Type II R–M systems comprise a separate restriction endonuclease (REase) and a methyltransferase (MTase) that act independently of each other. Type II REases generally recognize palindromic sequences in DNA and cleave within or near their recognition sequences and produce DNA fragments of defined sizes. They have become indispensable tools in molecular biology and have been widely exploited for studying site-specific protein–DNA interactions. Surprisingly, these enzymes share little or no sequence homology among them, though the three-dimensional structures determined to date reveal a common-core motif (‘PD...D/EXK’ motif) with a central β-sheet that is flanked by α-helices on both sides. In the motif, two acidic residues (D and D/E) are important for the metal ion binding and catalysis. The work presented in this thesis deals with the determination of active site, elucidation of kinetic mechanism and study of evolution of sequence specificity using the well known, R.KpnI, from Klebsiella pneumoniae. The enzyme is a homodimer, which recognizes a palindromic double stranded DNA sequence, GGTAC↓C, and cleaves as shown. Unlike other REases, R.KpnI shows prolific promiscuous DNA cleavage in presence of Mg2+. Surprisingly, Ca2+ completely suppresses the Mg2+ mediated promiscuous activity and induces high fidelity cleavage at the recognition sequence. These unusual properties of R.KpnI led to the characterization of the active site of the enzyme. This thesis is divided into five chapters. Chapter 1 is a general introduction of R-M systems and an overview of the literature on active sites of Type II REases. It deals with discovery, nomenclature and classification followed by description of the enzymes diversity and general features of Type II REases. The different active site folds of the REases have been discussed in detail. The features of sequence specificity and the efforts undertaken to engineer the new specificity in the REases have been dealt at the end of the chapter. Chapter 2 describes identification and characterization of the R.KpnI active site by bioinformatics analyses, homology modeling and mutational studies. Bioinformatics analyses reveal that R.KpnI contains a ββα-Me-finger fold, which is a characteristic of many HNH-superfamily endonucleases. According to the homology model of R.KpnI, the putative active site residues correspond to the conserved residues present in HNH nucleases. Substitutions of these conserved residues in R.KpnI resulted in loss of the DNA cleavage activity, confirming their importance. This study provides the first experimental evidence for a Type IIP REase that is a member of the HNH superfamily and does not belong to the PD...D/EXK superfamily of nucleases. In Chapter 3 DNA binding and kinetic analysis of R.KpnI is presented. The metal ions which exhibit disparate pattern of DNA cleavage have no role in DNA recognition. The enzyme binds to both canonical and non-canonical DNA with comparable affinity irrespective of the metal ions used. Further, it was shown that Ca2+-imparted exquisite specificity of the enzyme is at the level of DNA cleavage and not at the binding step. The kinetic constants were obtained through steady-state kinetic analysis of R.KpnI in presence of different metal ions. With the canonical oligonucleotides, the cleavage rate of the enzyme was comparable for both Mg2+- and Mn2+-mediated reactions and was about three times slower with Ca2+. The enzyme discriminates non-canonical sequences poorly from the canonical sequence in Mg2+-mediated reactions unlike any other Type II REases, accounting for its promiscuous behavior. These studies suggest that R.KpnI displays properties akin to that of typical Type II REases and also endonucleases with degenerate specificity for DNA recognition and cleavage. In chapter 4, two uncommon roles for Zn2+ in R.KpnI are described. Examination of the sequence revealed the presence of a zinc finger (CCCH) motif rarely found in proteins of prokaryotic origin. Biophysical experiments and subsequent mutational analysis showed that the zinc binding motif tightly coordinates zinc to provide a rigid structural framework for the enzyme needed for its function. In addition to this structural scaffold, another atom of zinc binds to the active site to induce high fidelity cleavage and suppress the Mg2+- and Mn2+-mediated promiscuous behavior of the enzyme. This is the first demonstration of distinct structural and catalytic roles for zinc in a REase. Chapter 5 describes generation of highly sequence specific R.KpnI. Towards this end, site-directed mutants were generated at the putative secondary metal binding site. The DNA binding and cleavage analyses of the mutants at putative secondary metal binding site revealed that the secondary site is not important for primary catalysis and have a role in sequence specificity. A single amino acid change at the D163 position abolished the promiscuous activity of the wt enzyme in the presence of Mg2+ and Mn2+. Thus, a single point mutation converts the promiscuous endonuclease to a high fidelity REase. In conclusion, the work described in the thesis reveals new information on the REases in general and R.KpnI in particular. Many of the properties of R.KpnI elucidated in this thesis represent hitherto unknown features amongst REases. The presence of an HNH catalytic motif in the enzyme indicates the diversity of active site fold in REases and their distinct origin. Similarly, the high degree of promiscuity exhibited by the enzyme may hint at the evolutionary link between non-specific and highly sequence specific nucleases. The present studies also provide an example for the role of mutations in the evolution of sequence specificity. The utilization of different metal ions for DNA cleavage and the architectural role for Zn2+ in maintaining the structural integrity are other unusual properties of the enzyme.

No description available.

The concentrations of trace elements in 30 Saanich Peninsula agricultural soil samples were determined by acid digestion and inductively coupled plasma - optical emission spectroscopy (ICP-OES). A comparison of the results obtained to a 1995 BC Ministry of Environment data indicated that As, Cu, Mo, Sb, Se and Sn concentrations had increased whereas the concentrations of Ba, Be, Cd, Cr, Co, Mn, Ni, Pb, V and Zn had decreased. The principal sources of the trace elements were anthropogenic sources including fertilizer and manure application, weathering of rocks and atmospheric deposition. The concentrations changes were largely influenced by the individual properties of the elements, soil texture, soil organic matter and clay content. The mobility of the trace elements in the soils was mainly controlled by clay content and followed the order Cd> B >Mo> Cr> V> Zn> Se> Co> Cr> As> Ba> Sb> Mn> Ag, Be, Hg, Ni, Pb.

A variety of extractants were used to fractionate the trace metals Cd, Cu, Mn, Ni, Pb, and Zn in contaminated urban soils. The metals were mostly held in soil solid phases but not in soluble and exchangeable forms. Except for Mn, extractability of metals by selective chemicals was mainly a function of the contamination level. The speciation of these elements in water, 0.01 M CaCl2, and pH-adjusted water extra was calculated using the chemical equilibrium model MINEQL+. The free ions of Cd, Ni, and Zn were the predominant species in most of the water and 0.01 M CaCl2 extracts while PbCO30 was the main form of Pb. Organically complexed Cu accounted for over of the total dissolved Cu. The solubility of trace metals increased as solution pH was decreased. Metals Cd, Ni, Pb and Zn combined with Cl- at low pH (<5). Organically complexed Cu was shifted to Cu2+ when pH was decreased. / Except for Mn, the activities of the other metals in water, 0.01 M CaCl 2 extracts, and pH-adjusted water extracts were undersaturated with respect to established minerals in soils. MnHPO4 seems to be controlling the solubility of Mn in these extracts.

The work in this thesis demonstrates whether resins are better than conventional soil extractants at predicting plant metal concentrations, and whether low-cost soil treatments can effectively reduce metal content and concentrations in lettuce leaves. / Researchers have shown that ion exchange resins can simulate root metal uptake behaviour by acting as ion sinks. Chemical extractants on the other hand, generally do not behave in this manner. Thus, we would expect resins (in either bead or membrane form) to predict plant metal concentrations better than conventional extractants. For the first study, a procedure using anion exchange resin membranes (AEM) treated with either EDTA or DTPA chelators was chosen, since previous studies showed this method to be effective at predicting plant metal uptake, and practical for routine laboratory use. In addition, several conventional extractants were selected for comparison. / For the second part of the thesis, a study was undertaken to test the effectiveness of various food processing byproducts to stabilize metal contaminants in soils, and compare the results with those of a Na-based aluminosilicate (zeolite). (Abstract shortened by UMI.)

As a result of rapid growth in technology, efforts to remove pollutants from the natural environment have not been able to keep pace with the increasing amount of waste materials and a growing population that further aggravates the situation. This has resulted in the transformation of lakes and ponds into pollution depots. A pilot study for monitoring certain selected trace elements entering and leaving YorkPrairie Creek Pond, situated inside the campus of Ball State University, was undertaken with the view to establishing whether or not this pond has been polluted, like many lakes and ponds, by metal corrosion, engine exhaust gases, and runoff from fertilizers and streets.This investigation involved sampling pond waters at two sites, viz., Influx surface and Effluent surface. Samples were collected once daily at the same time over a total time period of 23 days. Each sample was analyzed, in duplicate, for the heavy metal cations iron, lead, and cadmium, and for the inorganic anions nitrate, orthophosphate, and chloride. Metals were analyzed by atomic absorption spectrophotometry (A. A. S.), while titrimetric and cotorimetric procedures were utilized for the anion analyses.Results have indicated that iron and chloride are the most abundant of all analytes. Precipitation tends to Increase concentrations of analytes in general, possibly due to increased runoff and the lowering of the pH of these waters; leading to increased elemental release into solution. The relative responses of the analyzed ion levels in pond influx and effluent waters have been shown to be similar although their levels were significantly different.Analyte levels were found to be generally higher in effluent than in Influx waters; only chloride was found to depart from this general behavior. This finding led to the conclusion that the selected Ions, with the exception of chloride, tend not to accumulate in the waters of the pond but rather to escape with its effluent. This conclusion is subject to modification by future researchers, to whom the author has recommended a more expanded sampling program that would include the surface and sediment of the pond's interior since the nature of the results (especially for Pb and Cd) would seem to indicate not only that the pond does not exhibit thorough mixing of its constituents (homogeneity) but also the presence of an active remobilization process within its interior.

Trace metal contamination of the air-soil-tree system was examined in southern Quebec, where acid deposition and tree dieback have been recorded in high elevation forests. Mn pollution was emphasized due to its large emission from gasoline combustion in Canada. Airborne Cu, Mn, V, and Zn showed higher concentrations than those reported for other remote locations. Significant fluctuation in Mn concentrations during the winter-spring season was explored by air mass back trajectory analysis. The study suggested that high Mn concentrations resulted from the atmospheric long-range transport from Canadian industrialized and metropolitan regions. Metal concentrations in podzolic topsoils were generally higher than their world-wide average values. Concentrations of trace metals in balsam fir (Abies balsamea (L.) Mill) needles were below their suggested potential phytotoxic levels, except for Mn, which also increased with elevation. The scanning of needles with micro-PIXE showed no significant correlation between metal accumulation and epistomatal distribution on foliar surfaces. / Pathways of trace metals deposited in the soil-tree system were elucidated through application of $ sp{54}$Mn and $ sp{65}$Zn on shoot, bark, and soil surfaces in growth-chamber experiments with balsam fir seedlings. Uptake and accumulation by seedlings 70 days after application on the shoot surface was about 25-30% of the remaining activities for $ sp{54}$Mn and $ sp{65}$Zn. Less than 1% of absorbed isotopes was translocated from the bark surface to other plant organs, whereas more than 50% of the radioisotopes absorbed at the shoot moved to the rest of the seedling. Acidic wetness facilitated the metal absorption through tree surfaces. Downward movement of the radioisotopes in podzolic soils was documented, and accumulation in seedlings by root uptake was 5% of the remaining activity for $ sp{54}$Mn and 3% for $ sp{65}$Zn 70 days after application. No appreciable elemental migration from internal tissues to epicuticular wax layers was found, and the leaching ratio was below 0.5 and 1.0% for $ sp{54}$Mn and $ sp{65}$Zn, respectively. This study helps to understand the links between atmospheric deposition and the elevated levels of Mn in trees, and potential effects of acid deposition on the bioaccumulation of toxic metal pollutants in high elevation forests in southern Quebec.

Thesis (M.A.)--University of Hawaii at Manoa, 2005. / Includes bibliographical references (leaves 146-156). / Also available by subscription via World Wide Web / xxi, 156 leaves, bound ill., maps 29 cm

One of the first concerns for forensic anthropologists in dealing with skeletal remains in the Australian context is the determination of whether the remains are of anthropological, historical or archaeological interest. If fewer than 75 years have elapsed since death, remains are classified as anthropological and of forensic interest. However, an accurate and reliable method for estimating time since death (TSD) from human skeletal remains has thus far eluded forensic anthropologists. This study investigates the application in an Australian context of a novel approach proposed by Swift (2001) to dating skeletal remains from their contained levels of radioisotopes 210Po, 238U and 226Ra and trace elements. Radionuclide activity concentrations were determined using alpha and gamma spectrometry. Trace element concentrations were measured on three separate occasions using inductively coupled plasma mass spectrometry (ICP-MS). Discriminant analysis of the combination of activity concentration values for 210Po, 238U and 226Ra indicated the possibility of separation of bones derived from individuals who had died in the three eras of interest. Additionally, variations in the concentration levels of specific trace elements and certain inter-element relationships between elements also showed significant correlations with TSD. The study could not be exhaustive as access to human skeletal material was limited and additionally, the archaeological material had a different origin and post-death history to material from the more recent past. However, trend lines for inter-relationships between specific metals and for radionuclides indicated that all material fitted the same generally projected trends and as such, inferences with respect to variations of trace elements and radionuclides could be made with confidence. Bone radionuclide activity and calcium concentrations were all significantly higher in bones from the archaeological era than those from more recent eras, while trace lead concentrations contained in samples from the more recent historical era were significantly higher than those from other eras. Barium, lanthanum, rubidium, strontium, cerium and neodymium concentrations were all significantly correlated with one another and with radionuclide activity concentrations. Differences were found between the patterns of radionuclide activity and trace element concentrations between the skull and femur. The results of this study lend support to suggestions that multivariate analysis of trace element concentrations and radionuclide activity levels could aid in the estimation of time since death from skeletal remains in Australia. Although this study made use of only a limited amount of material, results clearly indicated the need to take into account variations arising from lifetime activities, diagenesis and bone type in applying the techniques to estimations of time since death. It highlights the need for a large-scale study using bone of known ages that systematically examines these influences on the estimation of time since death.

Mounted ill. / Offprint of the author's journal article in pocket / Includes bibliographies / xiii, 248 leaves, [50] leaves of plates, [3] folded leaves : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, 1985