Yeast Cell Lines Research Articles

Not Limited to E. coli: Versatile Expression Vectors for Mammalian Protein Expression.

Recombinant protein expression is not limited to E. coli or other prokaryotic systems. It is inevitable to use eukaryotic systems in order to express challenging mammalian proteins. Eukaryotic systems are able to perform complex posttranslational modifications like protein processing, phosphorylation, glycosylation, which are essential for stability and functionality of many proteins. Different eukaryotic protein expression systems employing yeast, insect, or mammalian cell lines are established with each having its own advantages and disadvantages. Often it is quite difficult to decide which will be the most optimal expression system as this depends highly on the protein itself. Expression in stable cell lines requires substantial screening of expressible constructs prior to developing a stable expression cell line. To achieve fast screening by transient expression in multiple hosts, versatile vectors can be applied. In this chapter, we present an overview of the most common multi-host vectors, which allow for fast expression analysis without tedious (re)cloning of the gene of interest in several different protein production systems. The protocols in this chapter describe the latest methods for fast transient expression in insect and mammalian cell lines.

Structure/Function Analysis of Recurrent Mutations in SETD2 Protein Reveals a Critical and Conserved Role for a SET Domain Residue in Maintaining Protein Stability and Histone H3 Lys-36 Trimethylation

The yeast Set2 histone methyltransferase is a critical enzyme that plays a number of key roles in gene transcription and DNA repair. Recently, the human homologue, SETD2, was found to be recurrently mutated in a significant percentage of renal cell carcinomas, raising the possibility that the activity of SETD2 is tumor-suppressive. Using budding yeast and human cell line model systems, we examined the functional significance of two evolutionarily conserved residues in SETD2 that are recurrently mutated in human cancers. Whereas one of these mutations (R2510H), located in the Set2 Rpb1 interaction domain, did not result in an observable defect in SETD2 enzymatic function, a second mutation in the catalytic domain of this enzyme (R1625C) resulted in a complete loss of histone H3 Lys-36 trimethylation (H3K36me3). This mutant showed unchanged thermal stability as compared with the wild type protein but diminished binding to the histone H3 tail. Surprisingly, mutation of the conserved residue in Set2 (R195C) similarly resulted in a complete loss of H3K36me3 but did not affect dimethylated histone H3 Lys-36 (H3K36me2) or functions associated with H3K36me2 in yeast. Collectively, these data imply a critical role for Arg-1625 in maintaining the protein interaction with H3 and specific H3K36me3 function of this enzyme, which is conserved from yeast to humans. They also may provide a refined biochemical explanation for how H3K36me3 loss leads to genomic instability and cancer.

Detection of Hepatitis C core antibody by dual-affinity yeast chimera and smartphone-based electrochemical sensing

Yeast cell lines were genetically engineered to display Hepatitis C virus (HCV) core antigen linked to gold binding peptide (GBP) as a dual-affinity biobrick chimera. These multifunctional yeast cells adhere to the gold sensor surface while simultaneously acting as a “renewable” capture reagent for anti-HCV core antibody. This streamlined functionalization and detection strategy removes the need for traditional purification and immobilization techniques. With this biobrick construct, both optical and electrochemical immunoassays were developed. The optical immunoassays demonstrated detection of anti-HCV core antibody down to 12.3pM concentrations while the electrochemical assay demonstrated higher binding constants and dynamic range. The electrochemical format and a custom, low-cost smartphone-based potentiostat ($20 USD) yielded comparable results to assays performed on a state-of-the-art electrochemical workstation. We propose this combination of synthetic biology and scalable, point-of-care sensing has potential to provide low-cost, cutting edge diagnostic capability for many pathogens in a variety of settings.

The New Era of Virus-Like Particles Based Vaccines

Virus-like particles or what is called VLPs for short are considered a useful tool in the development of vaccines. These VLPs-based vaccines have attracted the attention of many biotech and pharmaceutical companies as effective treatment for many types of non-infectious chronic diseases. VLPs simply resemble viruses; however they are noninfectious due to the lack of viral genome. The expression of envelop or capsid proteins result in the self-assembly of the particle, this assembly can easily be carried out in bacteria, yeast, plant cells or insect cell lines. The presence of repetitive viral surface protein epitopes is successful in eliciting strong B cell responses. VLPs have also high safety profile due to the lack of viral genome and lack of the ability to replicate. This area of research is not really new, the discovery of hepatitis B virus derived VLPs goes back to 1976.

Analysis of Protein Oligomeric Species by Sucrose Gradients.

Protein misfolding, aggregation, and accumulation are a common hallmark in various neurodegenerative diseases. Invariably, the process of protein aggregation is associated with both a loss of the normal biological function of the protein and a gain of toxic function that ultimately leads to cell death. The precise origin of protein cytotoxicity is presently unclear but the predominant theory posits that smaller oligomeric species are more toxic than larger aggregated forms. While there is still no consensus on this subject, this is a central question that needs to be addressed in order to enable the design of novel and more effective therapeutic strategies. Accordingly, the development and utilization of approaches that allow the biochemical characterization of the formed oligomeric species in a given cellular or animal model will enable the correlation with cytotoxicity and other parameters of interest.Here, we provide a detailed description of a low-cost protocol for the analysis of protein oligomeric species from both yeast and mammalian cell lines models, based on their separation according to sedimentation velocity using high-speed centrifugation in sucrose gradients. This approach is an adaptation of existing protocols that enabled us to overcome existing technical issues and obtain reliable results that are instrumental for the characterization of the types of protein aggregates formed by different proteins of interest in the context of neurodegenerative disorders.

Oral Rabies Vaccine Design for Expression in Plants.

Vaccination is the sensitization process of the immune system against any pathogen. Generally, recombinant subunit vaccines are considered safer than attenuated vaccines. As whole pathogenic organisms are used in the immunization process, the attenuated vaccines are considered more risky than subunit vaccines. Rabies is the oldest known zoonosis which spreads through a neurotropic Lyssavirus primarily mediated through infected canine bites. Rabies causes worldwide loss of more than 60,000 human lives every year. Animal vaccination is equally important to check the transmission of rabies into humans. Rabies oral vaccination can be a good alternative where multiple booster and priming regimens are required while the painful vaccination process can continue for long durations. Introduction of oral vaccines was made to ease the discomfort associated with the mode of introduction of conventional vaccines into the body. Although the rabies oral vaccine can substantially reduce the cost of vaccination in the developing countries, mass immunization programs need larger quantities of vaccines which should be delivered at nominal cost. Expression of recombinant antigen proteins in E. coli is often not viable because of lack of post-translational modifications and folding requirements. Though yeast and insect cell line expression systems have post-translational processing and modifications, significantly different immunological response against their post-translational modification pattern limits their deployment as an expression system. As an alternative, plants are emerging as a promising system to express and deliver wide range of functionally active biopharmaceutical product at lower cost for mass immunization programs. As generation of vaccine antigenic proteins in plant systems are cheaper, the strategy will benefit developing countries where this disease causes thousands of deaths every year. In this chapter, we will discuss about our efforts toward development of oral rabies vaccine and the methodological steps involved during this procedure in detail.

Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening

Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80–90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.

Filamentous Structure of Cytidine Triphosphate Synthase

Nucleotide biosynthesis is a highly regulated process essential for cell growth and replication. Cytidine triphosphate synthase (CTPsyn) is a metabolic enzyme that catalyzes the rate-limiting step of de novo CTP biosynthesis. In addition to being an essential nucleotide, CTP is a basic building molecule (or block) of RNA, DNA, and phospholipids. Recently, CTPsyn was independently revealed to have a filamentous structure in bacteria, budding yeast, Drosophila, and mammalian cell lines, indicating that its structure is highly evolutionarily conserved. In the present review, we briefly discuss recent findings and suggest that future studies investigating the biological effect of the polymeric structure of CTPsyn will be expected.

N-Myristoylation of the Rpt2 subunit of the yeast 26S proteasome is implicated in the subcellular compartment-specific protein quality control system

Ubiquitination is the posttranslational modification of a protein by covalent attachment of ubiquitin. Controlled proteolysis via the ubiquitin–proteasome system (\\UPS) alleviates cellular stress by clearing misfolded proteins. In budding yeast, UPS within the nucleus degrades the nuclear proteins as well as proteins imported from the cytoplasm. While the predominantly nuclear localization of the yeast proteasome is maintained by the importin-mediated transport, N-myristoylation of the proteasome subunit Rpt2 was indicated to cause dynamic nucleo-cytoplasmic localization of proteasomes. Here, we quantitatively analyzed the ubiquitinated peptides using anti-K-ε-GG antibody in yeast cell lines with or without a mutation in the N-myristoylation site of Rpt2 and detected upregulated ubiquitination of proteins with nucleo-cytoplasmic localizations in the mutant strains. Moreover, both the protein and ubiquitinated peptide levels of two Hsp70 family chaperones involved in the nuclear import of misfolded proteins, Ssa and Sse1, were elevated in the mutant strains, whereas levels of an Hsp70 family chaperone involved in the nuclear export, Ssb, were reduced. Taken together, our results indicate that N-myristoylation of Rpt2 is involved in controlled proteolysis via regulation of the nucleo-cytoplasmic localization of the yeast proteasome.

Bio-activities and phytochemical investigation of <i>Cnestis palala</i> (Lour.) Merr.

Background: In traditional medicines, the root of Cnestis palala was used for the treatment of stomach ache, malaria, urinary track disorders and snakebite. The seed was used for poisoning rat and stray dogs. However, the bioactivities and chemical constituents have not been reported. So, this will be the first report. Material and Methods: Biological investigations of C. palala extracts against bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Propionibacterium acnes, Escherichia coli, and Pseudomonas aeruginosa), fungi (Trichophyton mentagrophytes, Trichophyton rubrum and Microsporum gypseum), yeast (Candida albicans), tuberculosis (Mycobacterium tuberculosis), malaria (Plasmodium falciparum) and cancer cell lines (MCF-7 and HT-29) were evaluated. The chromatographic and spectroscopic techniques were used for the isolation and identification of pure compounds. Results: The results showed that the ethanol leaf and bark extracts were active against S. aureus and S. epidermidis. The hexane leaf and seed extracts and petroleum ether bark and root extracts showed strongly inhibition values against MCF-7. Moreover, the petroleum ether bark extract exhibited high inhibition value against HT-29. Seven compounds were isolated as β-sitosterol-glucoside (CP1), hydroquinone (CP2), β-sitosterol (CP3), mixture of fatty acids (CP4) and ethyl caffeate (CP5), scopoletin (CP6) and 2-nonenal (CP7). The CP2 was strongly active against S. aureus and S. epidermidis, the MIC was showed 30.10 µg/ml and 15.05 µg/ml and the MBC was showed 15 µg/ml and 7.5 µg/ml, respectively. Conclusion: These results suggested that C. palala is a great potential source of anti-microbial agents. Hence, this is the first study, which will be the database for chemical constituents and bioactivities of C. palala.

The role of new eudesmane-type sesquiterpenoid and known eudesmane derivatives from the red alga Laurencia obtusa as potential antifungal–antitumour agents

A new eudesmane sesquiterpenoid, eudesma-4(15),7-diene-5,11-diol (1) along with the known trinor-sesquiterene, teuhetenone (2), and a seco-eudesmane sesquiterpene, chabrolidione B (3), have been isolated from the Red Sea red alga Laurencia obtusa. The chemical structures were elucidated on the basis of extensive spectroscopic analysis. The antifungal and cytotoxic activities of the isolated metabolites were tested against several fungi, yeast and human mammary carcinoma cell line (MCF-7). Compounds 1 and 3 showed a much better activity [minimum inhibitory concentration (MIC): 2.9 μM] than that of amphotericin B (MIC: 4.6 μM). Interestingly, compound 2, the least active antifungal compound, retained the high anticancer activity against MCF-7 (22 μM) in comparison with cisplatin (59 μM), which was determined by employing lactate dehydrogenase assay. Compounds 1–3 are recorded here for the first time from algal flora. The chemotaxonomic importance of the isolated metabolites was discussed.

SUV3 helicase is required for correct processing of mitochondrial transcripts.

Mitochondrial gene expression is largely regulated by post-transcriptional mechanisms that control the amount and translation of each mitochondrial mRNA. Despite its importance for mitochondrial function, the mechanisms and proteins involved in mRNA turnover are still not fully characterized. Studies in yeast and human cell lines have indicated that the mitochondrial helicase SUV3, together with the polynucleotide phosphorylase, PNPase, composes the mitochondrial degradosome. To further investigate the in vivo function of SUV3 we disrupted the homolog of SUV3 in Drosophila melanogaster (Dm). Loss of dmsuv3 led to the accumulation of mitochondrial mRNAs, without increasing rRNA levels, de novo transcription or decay intermediates. Furthermore, we observed a severe decrease in mitochondrial tRNAs accompanied by an accumulation of unprocessed precursor transcripts. These processing defects lead to reduced mitochondrial translation and a severe respiratory chain complex deficiency, resulting in a pupal lethal phenotype. In summary, our results propose that SUV3 is predominantly required for the processing of mitochondrial polycistronic transcripts in metazoan and that this function is independent of PNPase.

Evaluation of the sensitivity of bacterial and yeast cells to cold atmospheric plasma jet treatments.

The focus of this research was first to determine the influence of the atmospheric plasma drive frequency on the generation of atomic oxygen species and its correlation with the reduction of bacterial load after treatment in vitro. The treatments were carried out using a helium-plasma jet source called PlasmaStream™. The susceptibility of multiple microbial cell lines was investigated in order to compare the response of gram-positive and gram-negative bacteria, as well as a yeast cell line to the atmospheric plasma treatment. It was observed for the source evaluated that at a frequency of 160 kHz, increased levels of oxygen-laden active species (i.e., OH, NO) were generated. At this frequency, the maximum level of bacterial inactivation in vitro was also achieved. Ex vivo studies (using freshly excised porcine skin as a human analog) were also carried out to verify the antibacterial effect of the plasma jet treatment at this optimal operational frequency and to investigate the effect of treatment duration on the reduction of bacterial load. The plasma jet treatment was found to yield a 4 log reduction in bacterial load after 6 min of treatment, with no observable adverse effects on the treatment surface. The gram-negative bacterial cell lines were found to be far more susceptible to the atmospheric plasma treatments than the gram-positive bacteria. Flow cytometric analysis of plasma treated bacterial cells (Escherichia coli) was conducted in order to attain a fundamental understanding of the mode of action of the treatment on bacteria at a cellular level. This study showed that after treatment with the plasma jet, E. coli cells progressed through the following steps of cell death; the inactivation of transport systems, followed by depolarization of the cytoplasmic membrane, and finally permeabilization of the cell wall.

Parkinson Disease Mutant E46K Enhances α-Synuclein Phosphorylation in Mammalian Cell Lines, in Yeast, and in Vivo

Although α-synuclein (α-syn) phosphorylation has been considered as a hallmark of sporadic and familial Parkinson disease (PD), little is known about the effect of PD-linked mutations on α-syn phosphorylation. In this study, we investigated the effects of the A30P, E46K, and A53T PD-linked mutations on α-syn phosphorylation at residues Ser-87 and Ser-129. Although the A30P and A53T mutants slightly affected Ser(P)-129 levels compared with WT α-syn, the E46K mutation significantly enhanced Ser-129 phosphorylation in yeast and mammalian cell lines. This effect was not due to the E46K mutant being a better kinase substrate nor due to alterations in endogenous kinase levels, but was mostly linked with enhanced nuclear and endoplasmic reticulum accumulation. Importantly, lentivirus-mediated overexpression in mice also showed enhanced Ser-129 phosphorylation of the E46K mutant compared to WT α-syn, thus providing in vivo validation of our findings. Altogether, our findings suggest that the different PD-linked mutations may contribute to PD pathogenesis via different mechanisms.

Single-cell analysis of aneuploidy events using yeast whole chromosome painting probes (WCPPs)

Aneuploidy is considered a widespread genetic variation in such cell populations as yeast strains, cell lines and cancer cells, and spontaneous changes in the chromosomal copy number may have implications for data interpretation. Thus, aneuploidy monitoring is essential during routine laboratory practice, especially while conducting biochemical and/or gene expression analyses. In the present study, we constructed a panel of whole chromosome painting probes (WCPPs) to monitor aneuploidy in a single yeast Saccharomyces cerevisiae cell. The WCPP-based system was validated using “normal” haploid and diploid cells, as well as disomic cells both with and without cell synchronisation. FISH that utilised WCPPs was combined with DNA cell cycle analysis (imaging cytometry) to provide a detailed analysis of signal variability during the cell cycle. Chromosome painting can be utilised to detect spontaneously formed disomic chromosomes and study aneuploidy-promoting conditions. For example, the frequency of disomic chromosomes was increased in cells lacking NAD+-dependent histone deacetylase Sir2p compared with wild-type cells (p<0.05). In conclusion, WCPPs may be considered to be a powerful molecular tool to identify individual genomic differences. Moreover, the WCPP-based system may be used at the single-cell level of analysis to supplement array-based techniques and high-throughput analyses at the population scale.

Tools and methods to analyze autophagy in C. elegans

For a long time, autophagy has been mainly studied in yeast or mammalian cell lines, and assays for analyzing autophagy in these models have been well described. More recently, the involvement of autophagy in various physiological functions has been investigated in multicellular organisms. Modification of autophagy flux is involved in developmental processes, resistance to stress conditions, aging, cell death and multiple pathologies. So, the use of animal models is essential to understand these processes in the context of different cell types and during the whole life. For ten years, the nematode Caenorhabditis elegans has emerged as a powerful model to analyze autophagy in physiological or pathological contexts. In this article, we present some of the established approaches and the emerging tools available to monitor and manipulate autophagy in C. elegans, and discuss their advantages and limitations.

Knowledge-fused differential dependency network models for detecting significant rewiring in biological networks.

BackgroundModeling biological networks serves as both a major goal and an effective tool of systems biology in studying mechanisms that orchestrate the activities of gene products in cells. Biological networks are context-specific and dynamic in nature. To systematically characterize the selectively activated regulatory components and mechanisms, modeling tools must be able to effectively distinguish significant rewiring from random background fluctuations. While differential networks cannot be constructed by existing knowledge alone, novel incorporation of prior knowledge into data-driven approaches can improve the robustness and biological relevance of network inference. However, the major unresolved roadblocks include: big solution space but a small sample size; highly complex networks; imperfect prior knowledge; missing significance assessment; and heuristic structural parameter learning.ResultsTo address these challenges, we formulated the inference of differential dependency networks that incorporate both conditional data and prior knowledge as a convex optimization problem, and developed an efficient learning algorithm to jointly infer the conserved biological network and the significant rewiring across different conditions. We used a novel sampling scheme to estimate the expected error rate due to “random” knowledge. Based on that scheme, we developed a strategy that fully exploits the benefit of this data-knowledge integrated approach. We demonstrated and validated the principle and performance of our method using synthetic datasets. We then applied our method to yeast cell line and breast cancer microarray data and obtained biologically plausible results. The open-source R software package and the experimental data are freely available at http://www.cbil.ece.vt.edu/software.htm.ConclusionsExperiments on both synthetic and real data demonstrate the effectiveness of the knowledge-fused differential dependency network in revealing the statistically significant rewiring in biological networks. The method efficiently leverages data-driven evidence and existing biological knowledge while remaining robust to the false positive edges in the prior knowledge. The identified network rewiring events are supported by previous studies in the literature and also provide new mechanistic insight into the biological systems. We expect the knowledge-fused differential dependency network analysis, together with the open-source R package, to be an important and useful bioinformatics tool in biological network analyses.

The mINO80 chromatin remodeling complex is required for efficient telomere replication and maintenance of genome stability

The INO80 (inositol requiring mutant 80) chromatin remodeling complex plays important roles in transcriptional regulation and DNA replication and repair, and consists of several functional protein subunits, including the critical Ino80 ATPase catalytic subunit. While the function of INO80 has been studied in yeast and mammalian cell lines, we do not know how mIno80 contributes to the maintenance of genome stability to prevent cancer development in mice. Here, we use a conditional knockout approach to explore the cellular and organismal functions of mIno80. Deletion of mIno80 results in profound cellular proliferative defects and activation of p21-dependent cellular senescence. While mIno80 is required for efficient repair of DNA double strand breaks, its depletion did not impact upon the formation of γ-H2AX and 53BP1 DNA damage foci, or the activation of the ATM-CHK2-dependent DNA damage response. mIno80 deletion inhibited the generation of single-strand DNA, resulting in defects in homology-directed DNA repair (HDR) at telomeres. Fragile telomeres were prominent in mIno80(Δ/Δ) MEFs, suggesting that chromatin remodeling is required for efficient telomere replication. mIno80(-/-) mouse embryos die early during embryogenesis, while conditional deletion of mIno80 in adult mice results in weight loss and premature death. In a p53(-/-) tumor-prone background, mIno80 haploinsufficiency favored the development of sarcomas. Our studies suggest that the mIno80 chromatin remodeling complex plays important roles in telomere replication, HDR-mediated repair of dysfunctional telomeres, and maintenance of genome stability.

Identification and Functional Analysis of Trypanosoma cruzi Genes That Encode Proteins of the Glycosylphosphatidylinositol Biosynthetic Pathway

Background Trypanosoma cruzi is a protist parasite that causes Chagas disease. Several proteins that are essential for parasite virulence and involved in host immune responses are anchored to the membrane through glycosylphosphatidylinositol (GPI) molecules. In addition, T. cruzi GPI anchors have immunostimulatory activities, including the ability to stimulate the synthesis of cytokines by innate immune cells. Therefore, T. cruzi genes related to GPI anchor biosynthesis constitute potential new targets for the development of better therapies against Chagas disease.Methodology/Principal Findings In silico analysis of the T. cruzi genome resulted in the identification of 18 genes encoding proteins of the GPI biosynthetic pathway as well as the inositolphosphorylceramide (IPC) synthase gene. Expression of GFP fusions of some of these proteins in T. cruzi epimastigotes showed that they localize in the endoplasmic reticulum (ER). Expression analyses of two genes indicated that they are constitutively expressed in all stages of the parasite life cycle. T. cruzi genes TcDPM1, TcGPI10 and TcGPI12 complement conditional yeast mutants in GPI biosynthesis. Attempts to generate T. cruzi knockouts for three genes were unsuccessful, suggesting that GPI may be an essential component of the parasite. Regarding TcGPI8, which encodes the catalytic subunit of the transamidase complex, although we were able to generate single allele knockout mutants, attempts to disrupt both alleles failed, resulting instead in parasites that have undergone genomic recombination and maintained at least one active copy of the gene.Conclusions/SignificanceAnalyses of T. cruzi sequences encoding components of the GPI biosynthetic pathway indicated that they are essential genes involved in key aspects of host-parasite interactions. Complementation assays of yeast mutants with these T. cruzi genes resulted in yeast cell lines that can now be employed in high throughput screenings of drugs against this parasite.

Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code

Structural and biochemical studies have demonstrated that p73, p63 and p53 recognize DNA with identical amino acids and similar binding affinity. Here, measuring transactivation activity for a large number of response elements (REs) in yeast and human cell lines, we show that p53 family proteins also have overlapping transactivation profiles. We identified mutations at conserved amino acids of loops L1 and L3 in the DNA-binding domain that tune the transactivation potential nearly equally in p73, p63 and p53. For example, the mutant S139F in p73 has higher transactivation potential towards selected REs, enhanced DNA-binding cooperativity in vitro and a flexible loop L1 as seen in the crystal structure of the protein–DNA complex. By studying, how variations in the RE sequence affect transactivation specificity, we discovered a RE-transactivation code that predicts enhanced transactivation; this correlation is stronger for promoters of genes associated with apoptosis.