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Informal learning environments play a critical role in science, technology, engineering, and mathematics learning across the lifespan and are consequential in informing public understanding and engagement. This can be difficult to accomplish in life science where expertise thresholds and logistics involved with handling biological materials can restrict access. Community laboratories are informal learning environments that provide access to the resources necessary to carry out pursuits using enabling biotechnologies. We investigate a group of these spaces in order to ascertain how this occurs—with specific attention to how material and intellectual resources are structured and shape learning. Using surveys and focus group interviews, we explore a group of these spaces located in the United States. We found that the spaces examined offer learning activities that are sufficiently scaffolded and flexible as to promote personalized and community-driven practice. We discuss these findings in relation to informal learning environment design and learning.

The COVID-19 pandemic has challenged undergraduate instructors and students in an unprecedented manner. Each has needed to find creative ways to continue the engaged teaching and learning process in an environment defined by physical separation and emotional anxiety and uncertainty. As a potential tool to meet this challenge, we developed a set of curricular materials that combined our respective life science teaching interests with the real-time scientific problem of the COVID-19 pandemic in progress. Discrete modules were designed that are engaging to students, implement active learning-based coursework in a variety of institutional and learning settings, and can be used either in person or remotely. The resulting interdisciplinary curriculum, dubbed "COVID-360," enables instructors to select from a menu of curricular options that best fit their course content, desired activities, and mode of class delivery. Here we describe how we devised the COVID-360 curriculum and how it represents our efforts to creatively and effectively respond to the instructional needs of diverse students in the face of an ongoing instructional crisis.

The skill of analyzing and interpreting research data is central to the scientific process, yet it is one of the hardest skills for students to master. While instructors can coach students through the analysis of data that they have either generated themselves or obtained from published articles, the burgeoning availability of preprint articles provides a new potential pedagogical tool. We developed a new method in which students use a cognitive apprenticeship model to uncover how experts analyzed a paper and compare the professional’s cognitive approach to their own. Specifically, students first critique research data themselves and then identify changes between the preprint and final versions of the paper that were likely the results of peer review. From this activity, students reported diverse insights into the processes of data presentation, peer review, and scientific publishing. Analysis of preprint articles is therefore a valuable new tool to strengthen students’ information literacy and understanding of the process of science.

Animal remains represent ephemeral resources that provide nutrients to a wide range of organisms. On death, vertebrate carrion is immediately colonized with a variety of microorganisms (typically obligate or facultatively anaerobic bacteria from the air, from insects, or from the corpse itself), which produce odors through the breakdown of tissues, the alteration of volatile chemicals present in the environment, or both. Within minutes, certain necrophagous flies are attracted by these chemical signals, resulting in waves of oviposition and larviposition activity. Although there are certainly detrimental (pathogenic) bacteria in the milieu, there is significant evidence suggesting that the presence of bacteria in or on the corpse seems to aid in larval development and pupariation. This may be because of a change in larval nutrition, with the bacteria either being used as a food source themselves or making nutrients more available to larvae. Maggots also produce and secrete or excrete antimicrobial molecules that are effective in killing certain bacteria. It is unclear whether this is a defensive mechanism, a selective measure to enhance the survival of bacteria beneficial to the larva, or a combination of both. Significant research is still needed to fully appreciate the potential role that these bacteria—insect interactions have in conferring a competitive advantage for surviving in a carrion community.

Synthetic biology projects aim to produce physical DNA that matches a designed target sequence. Chemically synthesized oligomers are generally used as the starting point for building larger and larger sequences. Due to the error rate of chemical synthesis, these oligomers can have many differences from the target sequence. As oligomers are joined together to make larger and larger synthetic intermediates, it becomes essential to perform quality control to eliminate intermediates with errors and retain only those DNA molecules that are error free with respect to the target. This step is often performed by transforming bacteria with synthetic DNA and sequencing colonies until a clone with a perfect sequence is identified. Here we present CloneQC, a lightweight software pipeline available as a free web server and as source code that performs quality control on sequenced clones. Input to the server is a list of desired sequences and forward and reverse reads for each clone. The server generates summary statistics (error rates and success rates target-by-target) and a detailed report of perfect clones. This software will be useful to laboratories conducting in-house DNA synthesis and is available at http://cloneqc.thruhere.net/ and as Berkeley Software Distribution (BSD) licensed source.

Yeast and mammalian genomes are replete with nearly identical copies of long dispersed repeats in the form of retrotransposons. Mechanisms clearly exist to maintain genome structure in the face of potential rearrangement between the dispersed repeats, but the nature of this machinery is poorly understood. Here we describe a series of distinct “retrotransposon overdose” (RO) lineages in which the number of Ty1 elements in the Saccharomyces cerevisiae genome has been increased by as much as 10 fold. Although these RO strains are remarkably normal in growth rate, they demonstrate an intrinsic supersensitivity to DNA-damaging agents. We describe the identification of mutants in the DNA replication pathway that enhance this RO-specific DNA damage supersensitivity by promoting ectopic recombination between Ty1 elements. Abrogation of normal DNA replication leads to rampant genome instability primarily in the form of chromosomal aberrations and confirms the central role of DNA replication accuracy in the stabilization of repetitive DNA.

Mobile elements are important components of our genomes, with diverse and significant effects on phenotype. Not only can transposons inactivate genes by direct disruption and shuffle the genome through recombination, they can also alter gene expression subtly or powerfully. Currently active transposons are highly polymorphic in host populations, including, among hundreds of others, L1 and Alu elements in humans and Ty1 elements in yeast. For this reason, we wished to develop a simple genome-wide method for identifying all transposons in any given sample. We have designed a transposon insertion site profiling chip (TIP-chip), a microarray intended for use as a high-throughput technique for mapping transposon insertions. By selectively amplifying transposon flanking regions and hybridizing them to the array, we can locate all transposons present in a sample. We have tested the TIP-chip extensively to map Ty1 retrotransposon insertions in yeast and have achieved excellent results in two laboratory strains as well as in evolved Ty1 high-copy strains. We are able to identify all of the theoretically detectable transposons in the FY2 lab strain, with essentially no false positives. In addition, we mapped many new transposon copies in the high-copy Ty1 strain and determined its Ty1 insertion pattern.

The retroviral Gag polyprotein orchestrates the assembly and release of virus particles from infected cells. We previously reported that nuclear transport of the Rous sarcoma virus (RSV) Gag protein is intrinsic to the virus assembly pathway. To identify cis- and trans- acting factors governing nucleocytoplasmic trafficking, we developed novel vectors to express regions of Gag in Saccharomyces cerevisiae . The localization of Gag proteins was examined in the wild type and in mutant strains deficient in members of the importin-β family. We confirmed the Crm1p dependence of the previously identified Gag p10 nuclear export signal. The known nuclear localization signal (NLS) in MA (matrix) was also functional in S. cerevisiae , and additionally we discovered a novel NLS within the NC (nucleocapsid) domain of Gag. MA utilizes Kap120p and Mtr10p import receptors while nuclear entry of NC involves the classical importin-α/β (Kap60p/95p) pathway. NC also possesses nuclear targeting activity in avian cells and contains the primary signal for the import of the Gag polyprotein. Thus, the nucleocytoplasmic dynamics of RSV Gag depend upon the counterbalance of Crm1p-mediated export with two independent NLSs, each interacting with distinct nuclear import factors.

The Rous sarcoma virus (RSV) Gag polyprotein undergoes transient nuclear trafficking as an intrinsic part of the virus assembly pathway. Nuclear export of Gag is crucial for the efficient production of viral particles and is accomplished through the action of a leptomycin B (LMB)-dependent nuclear export signal (NES) in the p10 domain (L. Z. Scheifele, R. A. Garbitt, J. D. Rhoads, and L. J. Parent, Proc. Natl. Acad. Sci. USA 99: 3944-3949, 2002). We have now mapped the nuclear export activity to the C-terminal portion of the p10 sequence and identified the four hydrophobic amino acids within this region that comprise a leucine-rich NES. Alteration of these hydrophobic residues resulted in the accumulation of Gag proteins within the nucleus and a budding defect greater than that obtained with LMB treatment of cells expressing the wild-type Gag protein (Scheifele et al., Proc. Natl. Acad. Sci. USA 99: 3944-3949, 2002). In addition, export of Gag from the nucleus was found to be a rate-limiting step in virus-like particle production. Consistent with a role for the NES sequence in viral replication, this cluster of hydrophobic residues in p10 is conserved across a wide range of avian retroviruses. Furthermore, naturally occurring substitutions within this region in related viruses maintained nuclear export activity and remained sensitive to the activity of LMB. Using gain-of-function approaches, we found that the hydrophobic motif in p10 was sufficient to promote the nuclear export of a heterologous protein and was positionally independent within the Gag polyprotein. Finally, the export pathway was further defined by the ability of specific nucleoporin inhibitors to prevent the egress of Gag from the nucleus, thereby identifying additional cellular mediators of RSV replication.

A major challenge in undergraduate life science curricula is the continual evaluation and development of courses that reflect the constantly shifting face of contemporary biological research. Synthetic biology offers an excellent framework within which students may participate in cutting-edge interdisciplinary research and is therefore an attractive addition to the undergraduate biology curriculum. This new discipline offers the promise of a deeper understanding of gene function, gene order, and chromosome structure through the de novo synthesis of genetic information, much as synthetic approaches informed organic chemistry. While considerable progress has been achieved in the synthesis of entire viral and prokaryotic genomes, fabrication of eukaryotic genomes requires synthesis on a scale that is orders of magnitude higher. These high-throughput but labor-intensive projects serve as an ideal way to introduce undergraduates to hands-on synthetic biology research. We are pursuing synthesis of Saccharomyces cerevisiae chromosomes in an undergraduate laboratory setting, the Build-a-Genome course, thereby exposing students to the engineering of biology on a genomewide scale while focusing on a limited region of the genome. A synthetic chromosome III sequence was designed, ordered from commercial suppliers in the form of oligonucleotides, and subsequently assembled by students into ∼750-bp fragments. Once trained in assembly of such DNA “building blocks” by PCR, the students accomplish high-yield gene synthesis, becoming not only technically proficient but also constructively critical and capable of adapting their protocols as independent researchers. Regular “lab meeting” sessions help prepare them for future roles in laboratory science.

Transposable elements are ubiquitous among sequenced genomes. The host genomes roughly subdivide into two types: 1) streamlined, that is, small,with little space between genes and lacking large introns, or 2) bulky, with lots of space between genes and many large introns. Most microorganisms, along with selected vertebrates like the pufferfish, fall into the first class, whereas mammals and most plants fall into the second class. As can be seen from Fig. 1, transposable element abundance mirrors the genome type of the host, with mobile elements comprising half or more of many of these bulky genomes! Mobile elements are of two basic types: DNA transposons, which predominantly mobilize via a cut and paste mechanism, and retrotransposons, which move by a copy and paste mechanism involving reverse transcription of an RNA intermediate (Fig. 1 right panel; Curcio and Derbyshire 2003). Retrotransposons are found in virtually all eukaryotes, from yeast (Kim et al. 1998) to human (Lander et al. 2001). Remarkably, in a yeast cell, the number of retrotransposon copies can be changed rather dramatically without a major impact on the phenotype of the host. The change in copy number can be seen using a new tiling array technique by which it is possible to comprehensively map the unique genomic regions adjacent to all transposable element copies probed (Fig. 2; Wheelan et al. 2006). The ability of yeast strains to tolerate very high copy numbers of transposons is due in part to the fact that, in yeast, most insertions are targeted to non-essential genomic regions, even though most of the genome is protein-coding (Chalker and Sandmeyer 1990; Devine and Boeke 1996; Ji et al. 1993; Zou et al. 1996). This property and many others suggest that retrotransposons are highly coevolved with their hosts.

Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affinity despite having a deletion of the fourth alpha-helix of the M domain. Examination of the mutant protein's subcellular distribution revealed that it was not localized to the plasma membrane but instead was mistargeted to intracytoplasmic membranes. Specific plasma membrane targeting was restored by the addition of myristate plus a single basic residue, by multiple basic residues, or by the heterologous hydrophobic membrane-binding domain from the cellular Fyn protein. These results suggest that the fourth alpha-helix of the RSV M domain promotes specific targeting of Gag to the plasma membrane, either through a direct interaction with plasma membrane phospholipids or a membrane-associated cellular factor or by maintaining the conformation of Gag to expose specific plasma membrane targeting sequences.

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

Ionizing radiation is an established source of chromosome aberrations (CAs). Although double-strand breaks (DSBs) are implicated in radiation-induced and other CAs, the underlying mechanisms are poorly understood. Here, we show that, although the vast majority of randomly induced DSBs in G(2) diploid yeast cells are repaired efficiently through homologous recombination (HR) between sister chromatids or homologous chromosomes, approximately 2% of all DSBs give rise to CAs. Complete molecular analysis of the genome revealed that nearly all of the CAs resulted from HR between nonallelic repetitive elements, primarily Ty retrotransposons. Nonhomologous end-joining (NHEJ) accounted for few, if any, of the CAs. We conclude that only those DSBs that fall at the 3-5% of the genome composed of repetitive DNA elements are efficient at generating rearrangements with dispersed small repeats across the genome, whereas DSBs in unique sequences are confined to recombinational repair between the large regions of homology contained in sister chromatids or homologous chromosomes. Because repeat-associated DSBs can efficiently lead to CAs and reshape the genome, they could be a rich source of evolutionary change.