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.

Untangling the Organizational Ambiguities in Supply Chain Risk Recognition, Assessment, and Response: The Role of Network Embeddedness

In this thesis, I focus on supply chain risk related ambiguity, which represents the ambiguities firms exhibit in recognizing, assessing, and responding to supply chain disruptions. I, primarily, argue that ambiguities associated with recognizing and responding to supply chain risk are information gathering and processing problems. Guided by the theoretical perspective of bounded rationality, I propose a typology of supply chain risk related ambiguity with four distinct dimensions. I, also, argue that the major contributor to risk related ambiguity is often the environment, specifically the web of suppliers. Hence, I focus on the characteristics of these supplier networks to examine the sources of ambiguity. I define three distinct elements of network embeddedness – relational, structural, and positional embeddedness – and argue that the ambiguity faced by a firm in appropriately identifying the nature or impacts of major disruptions is a function of these network properties. Based on a survey of large North American manufacturing firms, I found that the extent of the relational ties a firm has and its position in the network are significantly related to supply chain risk related ambiguity. However, this study did not provide any significant support for the hypothesized relationship between structural embeddedness and ambiguity. My research contributes towards the study of supply chain disruptions by using the idea of bounded rationality to understand supply chain risk related ambiguity and by providing evidence that the structure of supply chain networks influences the organizational understanding of and responses to supply chain disruptions.