A genetic selection method for expression products that induce apoptosis in adherent mammalian cell lines.

The process of apoptosis is carefully controlled in cells, and different cell types display different sensitivities to pro-apoptotic stimuli. The prospect of exploiting such differences for treatment of diseases such as cancer, via novel therapeutic agents, is extremely attractive. Therefore, genetic selections for novel expression products that kill cells may have considerable value. However, such selections are difficult to devise and perform because the selected cells do not grow. We developed a selection scheme designed to enrich for genetic agents that kill cells. The selection is based on detachment of apoptotic cultured mammalian cells from adherent monolayers. We characterized the properties of these detached cells (floating cells), and various aspects of the selection process. This selection method is potentially applicable to many mammalian cell lines.

Transcriptional transactivation by selected short random peptides attached to lexA-GFP fusion proteins.

BACKGROUND: Transcriptional transactivation is a process with remarkable tolerance for sequence diversity and structural geometry. In studies of the features that constitute transactivating functions, acidity has remained one of the most common characteristics observed among native activation domains and activator peptides. RESULTS: We performed a deliberate search of random peptide libraries for peptides capable of conferring transcriptional transactivation on the lexA DNA binding domain. Two libraries, one composed of C-terminal fusions, the other of peptide insertions within the green fluorescent protein structure, were used. We show that (i) peptide sequences other than C-terminal fusions can confer transactivation; (ii) though acidic activator peptides are more common, charge neutral and basic peptides can function as activators; and (iii) peptides as short as 11 amino acids behave in a modular fashion. CONCLUSIONS: These results support the recruitment model of transcriptional activation and, combined with other studies, suggest the possibility of using activator peptides in a variety of applications, including drug development work.

Enrichment during transdominant genetic experiments using a flow sorter.

BACKGROUND: Flow cytometry, in combination with retroviral expression libraries, is a powerful tool for genetic experimentation in mammalian cells. Expression libraries are transduced into cells engineered with a fluorescent reporter. Sorting for either bright or dim cells allows enrichment for specific inhibitors that alter reporter activity. This strategy has been used to isolate peptides and RNAs that either activate or suppress defined biochemical pathways. METHODS: Several variables contribute to the enrichment process: (1) the background of the fluorescence bioassay; (2) the mean fluorescence ratio between the induced and noninduced reporter cell populations; (3) the genetic penetrance, or strength, of the inhibitor; and (4) the multiplicity of infection (MOI). An experimental and theoretical analysis, including computer modeling, of these issues in the context of a mammalian cell bioassay was undertaken. RESULTS: MOI measurements were shown to be problematic. High MOI had little effect on enrichment early in the cycling process but a significant effect at later stages. Penetrance and background were critical throughout the process. Enrichments within about twofold of the theoretical maximum were observed. CONCLUSIONS: Caution should be exercised in MOI determination because of the danger of significant underestimation. High MOI is potentially advantageous early in the selection process but hinders enrichment in the later rounds. Modeling shows that MOI, assay background and clone penetrance are the principal variables that determine the success of transdominant selections by FACS.

Template selection during manipulation of complex mixtures by PCR.

PCR is ubiquitous in molecular biology. It is used to amplify single sequences from large genomes, or populations of sequences from complex mixtures such as cDNA libraries in mammalian cells. These cDNA libraries are often employed in subsequent labor-intensive experiments such as genetic screens, the outcome of which depends on library quality. The use of PCR to amplify diverse sequence populations raises important technical issues. One question is whether or not PCR is capable of maintaining population diversity, specifically with respect to template selection in the first rounds of the amplification process (i.e., the possibility that rare sequences in a complex mixture are lost because of amplification failure at the outset of the PCR). Here, we analyze the properties of PCR in the context of template selection in complex mixtures and show that it is an excellent method for preserving diversity.

Peptide inhibitors expressed in vivo.

Peptide inhibitors isolated from libraries either through genetic screens or binding assays have gained visibility in the past year - especially with the publication of four studies in model systems (two in yeast, two in Escherichia coli). These and other studies demonstrate that forward and reverse genetic experiments with peptides can be extremely efficient in validating candidate drug targets and in defining elements of biochemical pathways.

Transdominant genetic analysis of a growth control pathway.

Genetic selections that use proteinaceous transdominant inhibitors encoded by DNA libraries to cause mutant phenocopies may facilitate genetic analysis in traditionally nongenetic organisms. We performed a selection for random short peptides and larger protein fragments (collectively termed "perturbagens") that inhibit the yeast pheromone response pathway. Peptide and protein fragment perturbagens that permit cell division in the presence of pheromone were recovered. Two perturbagens were derived from proteins required for pheromone response, and an additional two were derived from proteins that may negatively influence the pheromone response pathway. Furthermore, three known components of the pathway were identified as probable perturbagen targets based on physical interaction assays. Thus, by selection for transdominant inhibitors of pheromone response, multiple pathway components were identified either directly as gene fragments or indirectly as the likely targets of specific perturbagens. These results, combined with the results of previous work [Holzmayer, T. A., Pestov, D. G. & Roninson, I. B. (1992) Nucl. Acids. Res. 20, 711-717; Whiteway, M., Dignard, D. & Thomas, D. Y. (1992) Proc. Natl. Acad. Sci. USA 89, 9410-9414; and Gudkov, A. V., Kazarov, A. R., Thimmapaya, R., Axenovich, S. A., Mazo, I. A. & Roninson, I. B. (1994) Proc. Natl. Acad. Sci. USA 91, 3744-3748], suggest that transdominant genetic analysis of the type described here will be broadly applicable.

Green fluorescent protein as a scaffold for intracellular presentation of peptides.

Peptide aptamers provide probes for biological processes and adjuncts for development of novel pharmaceutical molecules. Such aptamers are analogous to compounds derived from combinatorial chemical libraries which have specific binding or inhibitory activities. Much as it is generally difficult to determine the composition of combinatorial chemical libraries in a quantitative manner, determining the quality and characteristics of peptide libraries displayed in vivo is problematical. To help address these issues we have adapted green fluorescent protein (GFP) as a scaffold for display of conformationally constrained peptides. The GFP-peptide libraries permit analysis of library diversity and expression levels in cells and allow enrichment of the libraries for sequences with predetermined characteristics, such as high expression of correctly folded protein, by selection for high fluorescence.

mRNA turnover in yeast promoted by the MATalpha1 instability element.

The decay rates of eukaryotic transcripts can be determined by sequence elements within an mRNA. One example of this phenomenon is the rapid degradation of the yeast MATalpha1 mRNA, which is promoted by a 65 nt segment of its coding region termed the MATalpha1 instability element (MIE). The MIE is also capable of destabilizing the stable PGK1 transcript. To determine how the MIE accelerates mRNA turnover we examined the mechanism of degradation of the MATalpha1 transcript. These experiments indicated that the MATalpha1 mRNA was degraded by a deadenylation-dependent decapping reaction which exposed the transcript to 5'-->3' exonucleolytic digestion. Deletion of the MIE from the MATalpha1 mRNA decreased the rate at which this mRNA was decapped. In contrast, insertion of the MIE into the PGK1 transcript caused an increase in the rate of deadenylation of the resulting chimeric mRNA. These observations suggest that the MIE promotes rapid mRNA decay by increasing the rates of deadenylation and decapping, with its primary effect on mRNA turnover depending on additional features of a given transcript. These results also strengthen the hypothesis that deadenylation-dependent decapping is a common pathway of mRNA decay in yeast and indicate that an instability element within the coding region of an mRNA can effect nucleolytic events that occur at both the 5'- and 3'-ends of an mRNA.

An essential component of the decapping enzyme required for normal rates of mRNA turnover.

A major pathway of messenger RNA degradation in eukaryotic cells is initiated by shortening of the poly(A) tail, which, at least in yeast, triggers a decapping reaction, thereby exposing the mRNA to 5' --> 3' degradation. Decapping is the key step in this decay pathway because the transcript body is rapidly degraded following decapping. Accordingly, decapping is the site of numerous controls, including inhibition of decapping by the poly(A) tail and modulation of mRNA decapping rate by specific sequences. Moreover, a specialized decay pathway that degrades aberrant transcripts triggers rapid mRNA decapping independently of poly(A)-tail shortening. We have identified a yeast gene, termed DCP1, that encodes the decapping enzyme, or an essential component of a decapping complex. The protein Dcp1 is required for the normal decay of many unstable and stable yeast mRNAs, as well as mRNAs that are decapped independently of deadenylation. These results indicate that mRNA-specific rates of decapping, and thus decay, will result from differences in the interaction of the DCP1 decapping enzyme with individual transcripts.

Multiple functions for the poly(A)-binding protein in mRNA decapping and deadenylation in yeast.

The first step in the decay of many eukaryotic mRNAs is shortening of the poly(A) tail. In yeast, deadenylation leads to mRNA decapping and subsequent 5' --> 3' exonucleolytic degradation of the transcript body. We have determined that the major poly(A)-binding protein Pab1p plays at least two critical roles in this pathway. First, mRNAs in pab1 delta strains were decapped prior to deadenylation. This observation defines a new function for Pab1p as an inhibitor of mRNA decapping. Moreover, mutations that inhibit mRNA turnover suppress the inviability of a pab1 delta mutation, suggesting that premature mRNA decapping in pab1 delta strains contributes to cell death. Second, we find that Pab1p is not required for deadenylation, although in its absence poly(A) tail shortening rates are significantly reduced. In addition, in the absence of Pab1p, newly synthesized mRNAs had poly(A) tails longer than those in wild-type strains and showed an unexpected temporal delay prior to the initiation of deadenylation and degradation. These results define new and critical functions for Pab1p in the regulation of mRNA decapping and deadenylation, two important control points in the specification of mRNA half-lives. Moreover, these results suggest that Pab1p functions in additional phases of mRNA metabolism such as mRNP maturation.

A small segment of the MAT alpha 1 transcript promotes mRNA decay in Saccharomyces cerevisiae: a stimulatory role for rare codons.

Differences in decay rates of eukaryotic transcripts can be determined by discrete sequence elements within mRNAs. Through the analysis of chimeric transcripts and internal deletions, we have identified a 65-nucleotide segment of the MAT alpha 1 mRNA coding region, termed the MAT alpha 1 instability element, that is sufficient to confer instability to a stable PGK1 reporter transcript and that accelerates turnover of the unstable MAT alpha 1 mRNA. This 65-nucleotide element is composed of two parts, one located within the 5' 33 nucleotides and the second located in the 3' 32 nucleotides. The first part, which can be functionally replaced by sequences containing rare codons, is unable to promote rapid decay by itself but can enhance the action of the 3' 32 nucleotides (positions 234 to 266 in the MAT alpha 1 mRNA) in accelerating turnover. A second portion of the MAT alpha 1 mRNA (nucleotides 265 to 290) is also sufficient to destabilize the PGK1 reporter transcript when positioned 3' of rare codons, suggesting that the 3' half of the MAT alpha 1 instability element is functionally reiterated within the MAT alpha 1 mRNA. The observation that rare codons are part of the 65-nucleotide MAT alpha 1 instability element suggests possible mechanisms through which translation and mRNA decay may be linked.