To fuse or not to fuse: What is your purpose?

Since the dawn of time, or at least the dawn of recombinant DNA technology (which for many of today''s scientists is the same thing), investigators have been cloning and expressing heterologous proteins in a variety of different cells for a variety of different reasons. These range from cell biological studies looking at protein-protein interactions, post-translational modifications, and regulation, to laboratory-scale production in support of biochemical, biophysical, and structural studies, to large scale production of potential biotherapeutics. In parallel, fusion-tag technology has grown-up to facilitate microscale purification (pull-downs), protein visualization (epitope tags), enhanced expression and solubility (protein partners, e.g., GST, MBP, TRX, and SUMO), and generic purification (e.g., His-tags, streptag, and FLAG™-tag). Frequently, these latter two goals are combined in a single fusion partner. In this review, we examine the most commonly used fusion methodologies from the perspective of the ultimate use of the tagged protein. That is, what are the most commonly used fusion partners for pull-downs, for structural studies, for production of active proteins, or for large-scale purification? What are the advantages and limitations of each? This review is not meant to be exhaustive and the approach undoubtedly reflects the experiences and interests of the authors. For the sake of brevity, we have largely ignored epitope tags although they receive wide use in cell biology for immunopreciptation.     

What is wrong with replacement series?

Replacement series methods have been widely used in studies of plant competition. Recent work suggests that they are usually inadequate to assess competitive interactions and can be misleading. Response models relating yield per individual to the densities of the component species provide a methodology for answering many questions about mixtures and also focus attention on the need for clarity in defining terminology and interpreting results.     

What is wrong with intelligent design?

This article reviews two standard criticisms of creationism/intelligent design (ID)): it is unfalsifiable, and it is refuted by the many imperfect adaptations found in nature. Problems with both criticisms are discussed. A conception of testability is described that avoids the defects in Karl Popper's falsifiability criterion. Although ID comes in multiple forms, which call for different criticisms, it emerges that ID fails to constitute a serious alternative to evolutionary theory.     

What is a cladogram and what is not?

The origins and meanings of “cladogram” are reviewed. Traditionally, “cladogram” has been defined as a graphical representation of an empirical hypothesis of relationships among taxa, based on evidence from synapomorphies alone. Disturbingly, numerous recent authors treat “cladogram” as synonymous with “dendrogram” and do not appreciate the particular methodological connotations of the former term. This is lamented.     

What is wrong with absolute individual fitness?

One of the most basic facts about evolution is that fitness is a relative concept. It does not matter how well an organism survives and reproduces, only that it does so better than other organisms bearing alternative traits. Nevertheless, many evolutionary arguments are framed in terms of absolute individual fitness. The absolute fitness criterion (AFC) can be justified in terms of relative fitness only given certain assumptions that are frequently violated in nature. In particular, interactions must occur in groups that are randomly formed and phenotypic variation among groups must be tightly coupled to genetic variation. Complicating the genotype-phenotype relationship can cause phenotypic variation among groups to become nonrandom, even when the groups are randomly formed, favoring traits that do not maximize absolute individual fitness. Complex genotype-phenotype relationships and complex population structures require explicit models of evolutionary change based on relative fitness differences within and among groups.     

What is wrong with Fanconi anemia cells?

Figuring out what is wrong in Fanconi anemia (FA) patient cells is critical to understanding the contributions of the FA pathway to DNA repair and tumor suppression. Although FA patients exhibit a wide range of disease manifestation as well as severity (asymptomatic to congenital abnormalities, bone marrow failure, and cancer), cells from FA patients share underlying defects in their ability to process DNA lesions that interfere with DNA replication. In particular, FA cells are very sensitive to agents that induce DNA interstrand crosslinks (ICLs). The cause of this pronounced ICL sensitivity is not fully understood, but has been linked to the aberrant activation of DNA damage repair proteins, checkpoints and pathways. Thus, regulation of these responses through coordination of repair processing at stalled replication forks is an essential function of the FA pathway. Here, we briefly summarize some of the aberrant DNA damage responses contributing to defects in FA cells, and detail the newly-identified relationship between FA and the mismatch repair protein, MSH2. Understanding the contribution of MSH2 and/or other proteins to the replication problem in FA cells will be key to assessing therapeutic options to improve the health of FA patients. Moreover, loss of these factors, if linked to improved replication, could be a key event in the progression of FA cells to cancer cells. Likewise, loss of these factors could synergize to enhance tumorigenesis or confer chemoresistance in tumors defective in FA-BRCA pathway proteins and provide a basis for biomarkers for disease progression and response.