Lipoamide dehydrogenase is a component of the multienzyme complexes
pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in Saccharomyces
cerevisiae, A mutant, Ipdl, lacking pyruvate dehydrogenase,
2-oxoglutarate dehydrogenase and lipoamide dehydrogenase activity had
previously been isolated in yeast. In addition, this mutant had been used
to screen a YEplS based yeast gene bank leading to the isolation of a
5,5 kb region of yeast DIA containing a gene, designated LFD, which
complements the mutation,
In this thesis the DIIA sequence of 2.7 kb of the 5,5 kb region is
presented. The sequenced region contains a 1.5 kb open reading frame
representing the LFD gene. From homology between the deduced amino acid
sequence of this open reading frame and the primary sequence of E. coli
and pig heart lipoamide dehydrogenase, the LFD gene has been shown to
represent the structural gene for lipoamide dehydrogenase. The primary
sequence encoded by the LPD gene also shows very strong homology to
several other related amino acid sequences including glutathione
reductase, mercuric reductase and the lipoamide dehydrogenase sequence
from A, vinlandii and human liver cells.
Analysis of the upstream region of the LFD gene led to the
identification of several sites of homology between sequences within this
region and known yeast regulatory motifs. These included three sites for
the binding of the GCI4 protein, a sequence very similar to the UAS2 of
CYC1, and three regions similar to the sequence TCACGTGA identified as an
important element within the promoter of the TFF1 and GAL2 genes in
S, cerevisiae, in the adenovirus major late promoter and as the binding
site of the centromere-binding protein, CPI.
Transcript analysis of LFD expression during conditions of amino
acid, starvation was carried out. In addition, gel retardation and BUasel
protection experiments, to investigate interactions between in vitro
synthesised GCI4 and the upstream region of the LFD gene, were performed.
The results from these experiments suggest but do not conclusively prove
that the LFD gene is subject to general amino acid control.
In an attempt to identify other DIA-binding proteins which interact
specifically with the LPD gene, protein fractions from an heparinSepharose column were assayed by gel retardation for binding to DIA
fragments from the 5' end of the gene. Several DIA-binding activities
were identified including a DIA-binding protein which binds regions
upstream and downstream of the translation start site. This DIA-binding
activity could be competed by the addition of increasing amounts of a
ligated oligonucleotide containing two copies of the TCACGTGA sequence.
The specificity and the regulatory role of the proteins identified
as binding to the 5' end of the LFD gene remain to be determined. In
this thesis a preliminary analysis of the elements involved the
regulation of this gene has been carried out and future directions, in the
study of transcriptional control of the LFD gene, discussed.