Co-expression and functional assay of human Rb and E2F1 proteins in yeast
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Date
1999-09
Authors
Wollenschlaeger, Alex
Journal Title
Journal ISSN
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Publisher
University of the Free State
Abstract
The mammalian cell cycle is composed of a myriad interactions occurring in a defined
sequence dictated by the flow of entropy. The decision to study the cell cycle requires
entry into a world where events take place, not because they want to, but because they have
to. Cells do not 'decide' to perform certain actions, but are driven by the laws of nature, the
same laws that are responsible for the existence of the universe. Study of this maelstrom of
reactions is truly analogous to opening Pandora's proverbial box. A peek inside and all the
inner workings of the cell start to spill out. Unfortunately, this is where things start to get
complicated.
Serendipitously, simpler alternatives are available. The yeast cell cycle is remarkably
similar to that of higher animals. The Rb and E2F 1 proteins are integral components of the
mammalian cell cycle, and as such, they are found in aberrant forms in numerous
malignancies. This necessitates an adequate means for the determination of the cellular
status of these proteins, as prognosis and diagnosis of several neoplastic disorders are
dependentthereon.
This study aimed to develop a yeast-based strategy for functional analysis of the human
tumour suppressor protein Rb. This goal was impeded by one factor; the yeast cell cycle is
too similar to that of humans. Several yeast strains, derived from W303-lA, were
constructed that each contain a reporter gene -the lacZ gene of E. coli- regulated by E2F
recognition elements introduced within the upstream CyC1 promoter. The theory was that
in the absence of Rb, ectopically expressed human E2F 1 protein would be able to bind the
RE and activate transcription of the reporter gene, ultimately resulting in a readily
observable product. In the presence of functional Rb, E2Fl would be bound by the tumour
suppressor protein, and thus be incapable of activating the reporter gene. This would
provide an assay of Rb status, based not on tedious sequencing analysis of the genetic
material, but on the actual functional activity of the protein.
When dealing with Mother Nature, though, we are oftentimes reminded that she has
thought of everything. S. cerevisiae contains an E2F-like activity, capable of binding the
exact RE introduced into the reporter gene promoter. This was confirmed by experiments
in this study, where the reporter gene was activated in the absence of ectopically expressed
E2F1 protein. The endogenous yeast E2F-like protein is thus able to activate transcription
of the reporter gene, negating the effect that would be observed by ectopic expression of
human E2F 1, and thus, all Rb-expression was performed in the absence of eo-expressed
human E2F 1. Since it is impossible to distinguish between the yeast and human E2F
activities, it is impossible to create a functional assay for Rb activity in the W303-1Aderived
chimeras constructed.
As mentioned previously, it could be possible to overcome this problem by knocking out
the yeast-borne E2F activity, but this approach is restricted by two barriers. Firstly, the
yeast equivalent of the E2F-family is yet to be cloned. This problem can be approached
with a transposon-based strategy. The endogenous E2F activity is capable of activating
transcription of the lacZ reporter gene, and in so doing, provides a convenient assay for
YE2F integrity. Through the use of a plasmid containing an inducible transposase it should
be possible to disrupt the YE2F -encoding gene through integration of a transposon. This
would be accompanied by an inability of the yeast to activate the reporter gene. The
transposon, containing flanking genomic DNA, could then be retrieved and provide the
basis for cloning the gene coding for YE2F. Since the reporter gene is specific for E2F-like
binding, retrieval of non-specific factors should be negated.
A second problem is that, since the E2F proteins play an important role in the progression
of the cell cycle in higher animals, it is possible that the yeast would not survive knocking
out its homologue. Relegation of YE2F to the role of bystander could possibly wreak
havoc with the delicate mechanism that is the cell cycle. Still, it would be interesting to
further pursue this idea.
Ectopic expression of human Rb in the various strains used in this study provided some
interesting results. Transformation of pAWl, followed by galactose-based induction of Rb
expression resulted in observable differences in growth characteristics of certain strains.
Those most affected were W-lf and W-1r, which each contain a single repeat of the E2F
RE within the upstream promoter of the reporter gene, albeit in forward and reverse
orientations, respectively. The effect was particularly evident in W-1r, where expression of
Rb resulted in complete cessation of growth, probably due to binding of yeast E2F-like
activity. These results could not, however, be reconciled with those obtained from cell
cycle analysis with the aid of flow cytometry. These experiments did not show any
significant effect of Rb expression on the cell cycle of the examined strains. This is
possibly due to an insufficient period of observation, but this could, unfortunately, neither
be confirmed nor dismissed.
From the results obtained in this study it appears that construction of an apposite reporter
system for the functional assay of Rb is perhaps more tricky than would be expected. The
interference of endogenous proteins is a cause for concern in a development strategy such
as this, and serves as a caveat for future studies in this field.
Description
Keywords
Cell cycle, Antioncogenes, Dissertation (M.Med.Sc. (Haematology and Cell Biology))--University of the Free State, 1999