Transaldolase is a key enzyme in the nonoxidative phase of the pentose phosphate pathway (PPP) which fulfills two essential functions, formation of ribose 5-phosphate for synthesis of nucleotides, RNA, and DNA and generation of NADPH for biosynthetic reactions and to maintain glutathione at a reduced state, thus, to protect sulfhydryl groups and cellular integrity from oxygen radicals. Importance of PPP in general, and of transaldolase in particular, have been demonstrated in host defense mechanisms against oxidative stress, embryogenesis, myelination, inflammation, lymphocyte activation, phagocytosis, cardiac arrhythmias, hepatomas, resistance to radiation of malignant tumors, and aging. Earlier studies indicated that tissue and disease-specific changes in transaldolase enzyme activity may be related to altered gene expression. However, progress into the mechanism of transaldolase expression has been hampered by a lack of reagents. We have cloned the human transaldolase (TAL-H) cDNA, the genomic locus, produced a functional recombinant protein, and generated specific antibodies. We have already identified two distinct transcriptional regulatory elements in the TAL-H locus. One is located upstream of the coding sequence while the second maps to a novel retrotransposon encompassed by exons 2 and 3 of the TAL-H gene. We will delineate, through deletion mapping and transfection experiments, regions of the gene required for tissue-specific expression and responses to regulatory signals. Precise boundaries of regulatory elements will be further characterized by in vitro transcription, gel retardation and footprinting studies. Transcription factors binding to the DAN sequence motifs will be identified by supershift and UV crosslinking analysis. Utilizing a 53 bp regulatory element from the retrotransposon region may allow isolation of novel DNA-binding proteins. The proposed experiments will permit to identify key factors controlling expression of TAL-H and delineate points of regulatory impact for PPP and the related metabolic, developmental, and disease processes.
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