Abstract
The overall objectives of this thesis were the isolation and expression of cDNA sequences coding for mammalian SHMT. A probe was developed using the available amino acid sequence of the rabbit cytosolic SHMT (Martini et al., 1987) as a basis for designing primers, which were then used to amplify a region of the sequences coding for rabbit cytosolic SHMT (chapter 3). This amplified region was subsequently used to screen a rabbit liver cDNA library. The screening proved successful, with the isolation of sixty-seven clones potentially coding for SHMT. Further analysis of one of these clones, pUS1203, has provided the nucleotide sequence of the complete coding sequences for cytosolic SHMT (chapter 4). Contained within this clone were the 5' and 3' UTRs of 155 and 653 nucleotides respectively. The complete SHMT cDNA was cloned into the expression vector pUS1000, to generate pUS1202, where transcription of the SHMT cDNA was driven by the human cytomegalovirus promoter. Expression of this recombinant SHMT in COS-1 cells was achieved (chapter 5). Site specific mutagenesis to remove an upstream ATG codon within the 5' UTR was found to dramatically increase the levels of SHMT activity, when the mutated SHMT cDNA (pUS1208) was transfected into COS-1 cells. The level of SHMT activity in the cells transfected with pUS1208 was 100-fold higher than the activity found in cells transfected with pUS1000 and 50-fold higher than in cells transfected with pUS1202. Subsequent screening of a human breast cancer cell line cDNA library with the complete rabbit SHMT cDNA insert in pUS1203, identified 20 possible clones containing SHMT sequence (chapter 6). Sequence analysis of the largest of these, pUS1206, revealed that it contained 930bp of SHMT coding sequences, 890bp of 3' UTR and 850bp of an intron located 5' to the coding sequences. Comparison of the human and rabbit SHMT cDNAs reveals a high degree of constraint for the coding sequences that extends to a region of the 3' UTR (chapter 7).