title: Biochemical Characterisation of Reconstructed Ancestral CM-DAH7PS Enzymes

authors: McMillan, Joel Patrick

subjects: DAH7PS

abstract: Thermophily has been proposed to be a primitive trait (Stetter, 2006, Pace, 1991, Woese, 1987) which has led to suggestions that all contemporary thermophilic species are the direct descendants of ancient thermophilic organisms. Ancestral sequence reconstruction (ASR) is a modern molecular technique which has been used to study the evolution of thermophily, however these studies have produced conflicting results. Studies utilising ASR to investigate the evolution of thermophily with two different proteins, elongation factor Tu and thioredoxin, have suggested a primitive origin for thermophily. However a recent study by Hobbs et al. (2012), in which ancestral IPMDH enzymes were reconstructed and biochemically characterised in order to investigate the evolution of thermophily in the Bacillus genus, suggested that thermophily may have evolved at least twice within the Bacillus genus, indicating that thermophily may not be strictly a primitive trait. This finding needs to be supported by data from other reconstructed enzymes.

Four ancestral Bacillus 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes with chorismate mutase fused to their N-termini (CM-DAH7PS) were reconstructed to investigate whether a similar trend would be observed with a different enzyme. The ages of these ancestral Precambrian enzymes were estimated to be between 570 and 1,079 million years old. The three most recent ancestral enzymes were characterised and deemed biologically feasible as evidenced by the kinetic properties they display, which are similar to those displayed by two extant CM-DAH7PS enzymes from Bacillus subtilis and Bacillus stearothermophilus. The X-ray crystal structure of the most recent CM-DAH7PS ancestor has been solved and found to be very similar to previously solved DAH7PS structures. The optimal temperatures for activity of the ancestral DAH7PS enzymes were used to infer how thermophily may have evolved in the Bacillus genus. Interestingly, the results conflict with the findings of Hobbs et al. (2012). This suggests that the biochemical characteristics of ancestral enzymes may be highly susceptible to the biochemical properties of the contemporary species used in the inference or that different enzymes may result in different inferences being drawn regarding the evolution of thermophily.

text: Biochemical Characterisation of Reconstructed Ancestral CM-DAH7PS Enzymes Thermophily has been proposed to be a primitive trait (Stetter, 2006, Pace, 1991, Woese, 1987) which has led to suggestions that all contemporary thermophilic species are the direct descendants of ancient thermophilic organisms. Ancestral sequence reconstruction (ASR) is a modern molecular technique which has been used to study the evolution of thermophily, however these studies have produced conflicting results. Studies utilising ASR to investigate the evolution of thermophily with two different proteins, elongation factor Tu and thioredoxin, have suggested a primitive origin for thermophily. However a recent study by Hobbs et al. (2012), in which ancestral IPMDH enzymes were reconstructed and biochemically characterised in order to investigate the evolution of thermophily in the Bacillus genus, suggested that thermophily may have evolved at least twice within the Bacillus genus, indicating that thermophily may not be strictly a primitive trait. This finding needs to be supported by data from other reconstructed enzymes.

Four ancestral Bacillus 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase enzymes with chorismate mutase fused to their N-termini (CM-DAH7PS) were reconstructed to investigate whether a similar trend would be observed with a different enzyme. The ages of these ancestral Precambrian enzymes were estimated to be between 570 and 1,079 million years old. The three most recent ancestral enzymes were characterised and deemed biologically feasible as evidenced by the kinetic properties they display, which are similar to those displayed by two extant CM-DAH7PS enzymes from Bacillus subtilis and Bacillus stearothermophilus. The X-ray crystal structure of the most recent CM-DAH7PS ancestor has been solved and found to be very similar to previously solved DAH7PS structures. The optimal temperatures for activity of the ancestral DAH7PS enzymes were used to infer how thermophily may have evolved in the Bacillus genus. Interestingly, the results conflict with the findings of Hobbs et al. (2012). This suggests that the biochemical characteristics of ancestral enzymes may be highly susceptible to the biochemical properties of the contemporary species used in the inference or that different enzymes may result in different inferences being drawn regarding the evolution of thermophily.

year: 2012

title: Insights into the experiences of older workers and change: Through the lens of selection, optimization and compensation

authors: Unson, Christine

subjects: encore

abstract: Purpose: The study examined the barriers faced, the goals selected, and the optimization and compensation strategies of older workers in relation to career change. Method: Thirty open-ended interviews, 12 in the United States and 18 in New Zealand, were conducted, recorded, transcribed verbatim, and analyzed for themes. Results: Barriers to finding and maintaining work included task difficulty, problems with coworkers and management, lack of self-confidence, health/physical limitations, ageism, and stereotyping. Respondents’ most frequently selected goals for a new career were the desire to help others, work satisfaction, and acquisition of learning. Seventy-five percent of respondents in paid employment had earning an income as a goal. Optimization strategies included drawing on past experience, assessing skills, seeking careers/organizations that fitted their values, and a willingness to accept lower pay or unpaid work. Attitudinal strategies included focus and persistence, optimism, and positive attitudes toward change. The compensation strategies reported were on-the-job training; help from coworkers, clients, or customers; friends and family social support; and professional networks. Management practices considered helpful were flexible work schedules, supervisors’ patience, and formal recognition of the value of older workers. Conclusion: This qualitative study, using the SOC framework, showed that evaluating one’s skills and values, being positive about change, and being part of a supportive work environment were key contributors to adaptive competence.

text: Insights into the experiences of older workers and change: Through the lens of selection, optimization and compensation Purpose: The study examined the barriers faced, the goals selected, and the optimization and compensation strategies of older workers in relation to career change. Method: Thirty open-ended interviews, 12 in the United States and 18 in New Zealand, were conducted, recorded, transcribed verbatim, and analyzed for themes. Results: Barriers to finding and maintaining work included task difficulty, problems with coworkers and management, lack of self-confidence, health/physical limitations, ageism, and stereotyping. Respondents’ most frequently selected goals for a new career were the desire to help others, work satisfaction, and acquisition of learning. Seventy-five percent of respondents in paid employment had earning an income as a goal. Optimization strategies included drawing on past experience, assessing skills, seeking careers/organizations that fitted their values, and a willingness to accept lower pay or unpaid work. Attitudinal strategies included focus and persistence, optimism, and positive attitudes toward change. The compensation strategies reported were on-the-job training; help from coworkers, clients, or customers; friends and family social support; and professional networks. Management practices considered helpful were flexible work schedules, supervisors’ patience, and formal recognition of the value of older workers. Conclusion: This qualitative study, using the SOC framework, showed that evaluating one’s skills and values, being positive about change, and being part of a supportive work environment were key contributors to adaptive competence.

year: 2012

title: Iwi interests and the RMA: An evaluation of the quality of first generation council plans

authors: Jefferies, Richard

subjects: New Zealand

abstract: This working paper analyses the processes adopted by councils for involving hapū
/iwi in plan implementation, including the resource consents process. Three topic issues were investigated to assess plan implementation — urban amenity,
storm water, and issues of importance to iwi. Questions were asked about the capacity of hapū/iwi to engage in the resource consent
process, which resource issues were of concern to them, their relationship with
council and consent applicants, and their perception of the consent process. Most resources listed in the questionnaire were of concern to hapū/iwi, with water
quality, wāhi tapu and heritage the most commonly cited. In conclusion, we found a general dissatisfaction on the part of hapū/iwi with
councils’ performance with respect to both Treaty relationships and consent
processing under the RMA. A further contributing factor to the poor relationships
found between hapū/iwi and councils, was the lack of clarity over the role of hapū and
iwi in resource management. In several districts, diverging responses from hapū/iwi
and councils to questions about level of understanding and commitment suggests there
is a need for more effective communication. These problems are compounded by the
generally low capacity of hapū/iwi to participate in resource consent processes. These findings suggest that there is much to be done to improve relationships and
behaviour of these key stakeholder groups in the plan implementation process if key
provisions in the RMA related to hapū/iwi interests are to be fulfilled. The differences
shown in reciprocal perceptions have serious implications for establishing a sound
working partnership between councils and hapū/iwi in their areas. Making clear these
discrepancies is a first step towards taking the measures needed for building a better
partnership. Further, the capacity of hapū/iwi to participate could be better utilised if
there was greater integration between regional and district councils on issues of
significance and processes for iwi involvement.

text: Iwi interests and the RMA: An evaluation of the quality of first generation council plans This working paper analyses the processes adopted by councils for involving hapū
/iwi in plan implementation, including the resource consents process. Three topic issues were investigated to assess plan implementation — urban amenity,
storm water, and issues of importance to iwi. Questions were asked about the capacity of hapū/iwi to engage in the resource consent
process, which resource issues were of concern to them, their relationship with
council and consent applicants, and their perception of the consent process. Most resources listed in the questionnaire were of concern to hapū/iwi, with water
quality, wāhi tapu and heritage the most commonly cited. In conclusion, we found a general dissatisfaction on the part of hapū/iwi with
councils’ performance with respect to both Treaty relationships and consent
processing under the RMA. A further contributing factor to the poor relationships
found between hapū/iwi and councils, was the lack of clarity over the role of hapū and
iwi in resource management. In several districts, diverging responses from hapū/iwi
and councils to questions about level of understanding and commitment suggests there
is a need for more effective communication. These problems are compounded by the
generally low capacity of hapū/iwi to participate in resource consent processes. These findings suggest that there is much to be done to improve relationships and
behaviour of these key stakeholder groups in the plan implementation process if key
provisions in the RMA related to hapū/iwi interests are to be fulfilled. The differences
shown in reciprocal perceptions have serious implications for establishing a sound
working partnership between councils and hapū/iwi in their areas. Making clear these
discrepancies is a first step towards taking the measures needed for building a better
partnership. Further, the capacity of hapū/iwi to participate could be better utilised if
there was greater integration between regional and district councils on issues of
significance and processes for iwi involvement.

year: 2002-10-31