An evaluation of the deputy principals\u27 leadership development program

The Deputy Principals\u27 Leadership Development Program (DPLDP) was researched and developed by the Calgary Board of Education from 1983 to 1987. It was conducted for the first time in Western Australia when deputy principals from schools in the Narrogin Education District participated in the program during the period 1989-91. This study is an evaluation of the DPLDP. It was designed to determine whether there is justification for the continued use of the program as a means of enhancing the instructional leadership capacities of deputy principals in the Narrogin Education District. This research is located within the naturalistic paradigm. It can best be described as a qualitative case study based mainly upon ethnographic methods of data collection. The organisation and analysis of the data, however, was structured largely by utilising Stufflebeam\u27s CIPP (context, input, process, product) framework of program evaluation and a typology of instructional leadership developed from a review of the literature. This typology presents instructional leadership as a process based on three components: vision, information and action. The data for this evaluation was collected during a twenty two month period between April 1989 and November 1990. A variety of data gathering techniques was used. In depth, semi-structured interviews and participant observations generated most of the data. Document analysis and unstructured, informal interactions provided supplementary material. Data validation formed an integral component of the research design. A thorough and wide-ranging validation process involving the participants was utilized to check the accuracy and relevance of the research findings. The major conclusions of the study, that emerged within the CIPP framework of program evaluation and the typology of instructional leadership, were: 1. Participation in the DPLDP leads deputy principals to examine their role in schools, and builds a commitment to their role as instructional leaders. 2. The DPLDP has the potential, more than other available programs examined, to meet the professional development needs of deputy principals in key areas associated with instructional leadership. 3. Collegial support is the single most important element of the DPLDP for the development of deputy principals as instructional leaders. 4. The DPLDP can be implemented successfully in the Narrogin Education without significant modifications. Based on these results, and other subsidiary findings of the study, it was concluded that, on balance, there is justification for the continuation of the DPLDP in the Narrogin Education District

Purification and Characterisation of Benzaldehyde Dehydrogenase I from Acinetobacter calcoaceticus and the TOL Plasmid Encoded Benzaldehyde Dehydrogenase and Benzyl Alcohol Dehydrogenase from Pseudomonas putida

1 Acinetobacter calcoaceticus can grow on either mandelate or benzyl alcohol as sole sources of carbon and energy. L(+)-Mandelate is metabolised to benzoate with phenylglyoxylate and benzaldehyde as intermediates, whereas benzyl alcohol is converted to benzoate with benzaldehyde as the only intermediate. Although the intermediates of the mandelate and benzyl alcohol pathways converge at the level of benzaldehyde, the enzymes of the pathways are quite separate because of the presence of two different benzaldehyde dehydrogenases. This thesis is concerned primarily with the purification and characterisation of benzaldehyde dehydrogenase I which is induced during growth on mandelate. This was intended to complete the characterisation of all of the enzymes of the mandelate and benzyl alcohol pathways in A. calcoaceticus. The project was subsequently enlarged to include the purification and characterisation of the benzaldehyde dehydrogenase and the benzyl alcohol dehydrogenase encoded by the TOL plasmid pWW53 in Pseudomonas putida MT53, because this would allow a more broadly-based comparison of five aldehyde and alcohol dehydrogenases, including a comparison of chromosomal and plasmid encoded enzymes

Base-flipping dynamics in a DNA hairpin processing reaction

Many enzymes that repair or modify bases in double-stranded DNA gain access to their substrates by base flipping. Although crystal structures provide stunning snap shots, biochemical approaches addressing the dynamics have proven difficult, particularly in complicated multi-step reactions. Here, we use protein–DNA crosslinking and potassium permanganate reactivity to explore the base-flipping step in Tn5 transposition. We present a model to suggest that base flipping is driven by a combination of factors including DNA bending and the intrusion of a probe residue. The forces are postulated to act early in the reaction to create a state of tension, relieved by base flipping after cleavage of the first strand of DNA at the transposon end. Elimination of the probe residue retards the kinetics of nicking and reduces base flipping by 50%. Unexpectedly, the probe residue is even more important during the hairpin resolution step. Overall, base flipping is pivotal to the hairpin processing reaction because it performs two opposite but closely related functions. On one hand it disrupts the double helix, providing the necessary strand separation and steric freedom. While on the other, transposase appears to position the second DNA strand in the active site for cleavage using the flipped base as a handle

The roles of the human SETMAR (Metnase) protein in illegitimate DNA recombination and non-homologous end joining repair

SETMAR is a fusion between a SET-domain methyltransferase gene and a mariner-family transposase gene, which is specific to anthropoid primates. However, the ancestral SET gene is present in all other mammals and birds. SETMAR is reported to be involved in transcriptional regulation and a diverse set of reactions related to DNA repair. Since the transcriptional effects of SETMAR depend on site-specific DNA binding, and are perturbed by inactivating the methyltransferase, we wondered whether we could differentiate the effects of the SET and MAR domains in DNA repair assays. We therefore generated several stable U2OS cell lines expressing either wild type SETMAR or truncation or point mutant variants. We tested these cell lines with in vivo plasmid-based assays to determine the relevance of the different domains and activities of SETMAR in DNA repair. Contrary to previous reports, we found that wild type SETMAR had little to no effect on the rate of cell division, DNA integration into the genome or non-homologous end joining. Also contrary to previous reports, we failed to detect any effect of a strong active-site mutation that should have knocked out the putative nuclease activity of SETMAR

Compensating for over-production inhibition of the Hsmar1 transposon in Escherichia coli using a series of constitutive promoters

© 2020 The Author(s). Background: Transposable elements (TEs) are a diverse group of self-mobilizing DNA elements. Transposition has been exploited as a powerful tool for molecular biology and genomics. However, transposition is sometimes limited because of auto-regulatory mechanisms that presumably allow them to cohabit within their hosts without causing excessive genomic damage. The papillation assay provides a powerful visual screen for hyperactive transposases. Transposition is revealed by the activation of a promoter-less lacZ gene when the transposon integrates into a non-essential gene on the host chromosome. Transposition events are detected as small blue speckles, or papillae, on the white background of the main Escherichia coli colony. Results: We analysed the parameters of the papillation assay including the strength of the transposase transcriptional and translational signals. To overcome certain limitations of inducible promoters, we constructed a set of vectors based on constitutive promoters of different strengths to widen the range of transposase expression. We characterized and validated our expression vectors with Hsmar1, a member of the mariner transposon family. The highest rate of transposition was observed with the weakest promoters. We then took advantage of our approach to investigate how the level of transposition responds to selected point mutations and the effect of joining the transposase monomers into a single-chain dimer. Conclusions: We generated a set of vectors to provide a wide range of transposase expression which will be useful for screening libraries of transposase mutants. The use of weak promoters should allow screening for truly hyperactive transposases rather than those that are simply resistant to auto-regulatory mechanisms, such as overproduction inhibition (OPI). We also found that mutations in the Hsmar1 dimer interface provide resistance to OPI in bacteria, which could be valuable for improving bacterial transposon mutagenesis techniques

A single active site in the mariner transposase cleaves DNA strands of opposite polarity

The RNase H structural fold defines a large family of nucleic acid metabolizing enzymes that catalyze phosphoryl transfer reactions using two divalent metal ions in the active site. Almost all of these reactions involve only one strand of the nucleic acid substrates. In contrast, cut-and-paste transposases cleave two DNA strands of opposite polarity, which is usually achieved via an elegant hairpin mechanism. In the mariner transposons, the hairpin intermediate is absent and key aspects of the mechanism by which the transposon ends are cleaved remained unknown. Here, we characterize complexes involved prior to catalysis, which define an asymmetric pathway for transpososome assembly. Using mixtures of wild-type and catalytically inactive transposases, we show that all the catalytic steps of transposition occur within the context of a dimeric transpososome. Crucially, we find that each active site of a transposase dimer is responsible for two hydrolysis and one transesterification reaction at the same transposon end. These results provide the first strong evidence that a DDE/D active site can hydrolyze DNA strands of opposite polarity, a mechanism that has rarely been observed with any type of nuclease

Cyclic changes in the affinity of protein–DNA interactions drive the progression and regulate the outcome of the Tn 10 transposition reaction

The Tn 10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn 10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions

Cyclic changes in the affinity of protein–DNA interactions drive the progression and regulate the outcome of the Tn10 transposition reaction

The Tn10 transpososome is a DNA processing machine in which two transposon ends, a transposase dimer and the host protein integration host factor (IHF), are united in an asymmetrical complex. The transitions that occur during one transposition cycle are not limited to chemical cleavage events at the transposon ends, but also involve a reorganization of the protein and DNA components. Here, we demonstrate multiple pathways for Tn10 transposition. We show that one series of events is favored over all others and involves cyclic changes in the affinity of IHF for its binding site. During transpososome assembly, IHF is bound with high affinity. However, the affinity for IHF drops dramatically after cleavage of the first transposon end, leading to IHF ejection and unfolding of the complex. The ejection of IHF promotes cleavage of the second end, which is followed by restoration of the high affinity state which in turn regulates target interactions

Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism

The bacterial Tn5 and Tn10 transposases have a single active site that cuts both strands of DNA at their respective transposon ends. This is achieved using a hairpin intermediate that requires the DNA to change conformation during the reaction. In Tn5 these changes are controlled in part by a flipped nucleoside that is stacked on a tryptophan residue in a hydrophobic pocket of the transposase. Here we have investigated the base flipping mechanism in Tn10 transposition. As in Tn5 transposition, we find that base flipping takes place after the first nick and is required for efficient hairpin formation and resolution. Experiments with an abasic substrate show that the role of base flipping in hairpin formation is to remove the base from the DNA helix. Specific interactions between the flipped base and the stacking tryptophan residue are required for hairpin resolution later in the reaction. We show that base flipping in Tn10 transposition is not a passive reaction in which a spontaneously flipped base is captured and retained by the protein. Rather, it is driven in part by a methionine probe residue that helps to force the flipped base from the base stack. Overall, it appears that base flipping in Tn10 transposition is similar to that in Tn5 transposition

Targeted DNA transposition in vitro using a dCas9-transposase fusion protein

Homology-directed genome engineering is limited by transgene size. Although DNA transposons are more efficient with large transgenes, random integrations are potentially mutagenic. Here we present an in vitro mechanistic study that demonstrates efficient Cas9 targeting of the mariner transposon Hsmar1. Integrations were unidirectional and tightly constrained to one side of the sgRNA binding site. Further analysis of the nucleoprotein intermediates demonstrated that the transposase and Cas9 moieties can bind their respective substrates independently or in concert. Kinetic analysis of the reaction in the presence of the Cas9 target–DNA revealed a delay between first and second strand cleavage at the transposon end. This step involves a significant conformational change that may be hindered by the properties of the interdomainal linker. Otherwise, the transposase moiety behaved normally and was proficient for integration in vitro and in Escherichia coli. Specific integration into the lacZ gene in E. coli was obscured by a high background of random integrations. Nevertheless, Cas9 is an attractive candidate for transposon-targeting because it has a high affinity and long dwell-time at its target site. This will facilitate a future optogenetic strategy for the temporal control of integration, which will increase the ratio of targeted to untargeted events