Primary structure and expression studies of the dihydrofolate reductase gene (DFRI) of Saccharomyces cerevisiae

The nucleotide sequence of a genomic DNA fragment thought previously to contain the dihydrofolate reductase gene (DFR1) of Saccharomyces cerevisiae by genetic criteria was determined. This DNA fragment of 1784' basepairs contains a large open reading frame from position 800 to 1432, which encodes a enzyme with a predicted molecular weight of 24,229.8 Daltons. Analysis of the amino acid sequence of this protein revealed that the yeast polypep·tide contained 211 amino acids, compared to the 186 residues commonly found in the polypeptides of other eukaryotes. The difference in size of the gene product can be attributed mainly to an insert in the yeast gene. Within this region, several consensus sequences required for processing of yeast nuclear and class II mitochondrial introns were identified, but appear not sufficient for the RNA splicing. The primary structure of the yeast DHFR protein has considerable sequence homology with analogous polypeptides from other organisms, especially in the consensus residues involved in cofactor and/or inhibitor binding. Analysis of the nucleotide sequence also revealed the presence of a number of canonical sequences identified in yeast as having some function in the regulation of gene expression. These include UAS elements (TGACTC) required for tIle amino acid general control response, and "TATA H boxes as well as several consensus sequences thought to be required for transcriptional termination and polyadenylation. Analysis of the codon usage of the yeast DFRl coding region revealed a codon bias index of 0.0083. this valve very close to zero suggestes 3 that the gene is expressed at a relatively low level under normal physiological conditions. The information concerning the organization of the DFRl were used to construct a variety of fusions of its 5' regulatory region with the coding region of the lacZ gene of E. coli. Some of such fused genes encoded a fusion product that expressed in E.coli and/or in yeast under the control of the 5' regulatory elements of the DFR1. Further studies with these fusion constructions revealed that the beta-galactosidase activity encoded on multicopy plasmids was stimulated transiently by prior exposure of yeast host cells to UV light. This suggests that the yeast PFRl gene is indu.ced by UV light and nlay in1ply a novel function of DHFR protein in the cellular responses to DNA damage. Another novel f~ature of yeast DHFR was revealed during preliminary studies of a diploid strain containing a heterozygous DFRl null allele. The strain was constructed by insertion of a URA3 gene within the coding region of DFR1. Sporulation of this diploid revealed that meiotic products segregated 2:0 for uracil prototrophy when spore clones were germinated on medium supplemented with 5-formyltetrahydrofolate (folinic acid). This finding suggests that, in addition to its catalytic activity, the DFRl gene product nlay play some role in the anabolisln of folinic acid. Alternatively, this result may indicate that Ura+ haploid segregants were inviable and suggest that the enzyme has an essential cellular function in this species.

Education in the halls : students' perspectives on cultural expression and resistance

This research is a qualitative study of cultural reproduction and resistance from students' perspectives. Thirteen teenagers (eight in attendance in regular high schools and five drop-outs) were recruited to take part and were involved to varying degrees through interviews, journal writing, and group interactive sessions. A purposive sampling design was used initially to recruit individuals known to the researcher through contacts in an alternate education setting. Other participants were recruited throughout the research phase. The theoretical aspects are premised on the work of Paul Willis, Michel Foucault, and Pierre Bourdieu. The reflexive praxeology of Bourdieu reflects the position taken as one way of understanding how students construct and respond to the situations of cultural dominance they experience in schools. The same reflexivity is offered for suggestions as to how teachers can respond to their own position in the education system.

The expression and development of teachers' capacities within two learning communities : a participant-observer case study

The learning community model has been an integral component of teacher development in Ontarian schools and beyond. This research was conducted to understand how teachers' personal capacity and professional, interpersonal, and organizational competencies are developed and expressed within this context. Nineteen elementary teachers and administrators participated in the study from November through January 2007. A qualitative case study methodology was used to investigate the role ofteachers' capacities and competencies in learning communities. Combined data sources from semistructured interviews, research journals, and document review were used to gather data about teachers' capacities and competencies. The study included 3 phases of analysis. In the final phase the analysis provided 3 qualities of the teachers at Jude and Mountain Schools (pseudonyms): identification as professionals, investment in others, and institutional affiliation that may explain how they differed from other educators. The data revealed these three themes, which provided an understanding of educators at Jude and Mountain Schools as dedicated professionals pushing practices to contribute to school life and address student learning needs, and as teachers who reflected on practices to continue expanding their skills. Teachers were heavily invested in creating a caring culture and in students' and team members' learning. Educators actively participated in solving problems and coplanning throughout the school levels and beyond, assumed collective responsibility for all pupils, and focused on generating school-wide consistent practices. These qualities and action patterns revealed teachers who invested time and effort in their colleagues, who committed to develop as professionals, and who affiliated closely with every aspect of school living.

Effects of social context on endocrine function and Zif268 expression in response to an acute stressor in adolescent and adult rats

There is a paucity of studies comparing social buffering in adolescents and adults, despite their marked differences in social behaviour. I investigated whether greater effects of social buffering on plasma corticosterone concentrations and expression of Zif268 in neural regions after an acute stressor would be found in adolescent compared with adult rats. Samples were obtained before and after one hour of isolation stress and after either one or three hours of recovery back in the colony with either a familiar or unfamiliar cage partner. Adolescent and adult rats did not differ in plasma concentrations of corticosterone at any time point. Corticosterone concentrations were higher after one hour isolation than at baseline (p < 0.001), and rats with a familiar partner during the recovery phase had lower corticosterone concentrations than did rats with an unfamiliar partner (p = 0.02). Zif268 immunoreactive cell counts were higher in the arcuate nucleus in both age groups after isolation (p = 0.007) and higher in the paraventricular nucleus of adolescents compared with adults during the recovery phase irrespective of partner familiarity. There was a significant decrease in immunoreactive cell counts after one hour isolation compared to baseline in the basolateral amygdala, central nucleus of the amygdala, and in the pyramidal layer of the hippocampus (all p < 0.05). An effect of partner familiarity on Zif268 immunoreactive cell counts was found in the granule layer of the dentate gyrus irrespective of age (higher in those with a familiar partner, p = 0.03) and in the medial prefrontal cortex in adolescents (higher with an unfamiliar partner, p = 0.02). Overall, the acute stress and partner familiarity produced a similar pattern of results in adolescents and adults, with both age groups sensitive to the social context.