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Experimental data about gene functions curated from the primary literature have enormous value for research scientists in understanding biology. Using the Gene Ontology (GO), manual curation by experts has provided an important resource for studying gene function, especially within model organisms. Unprecedented expansion of the scientific literature and validation of the predicted proteins have increased both data value and the challenges of keeping pace. Capturing literature-based functional annotations is limited by the ability of biocurators to handle the massive and rapidly growing scientific literature. Within the community-oriented wiki framework for GO annotation called the Gene Ontology Normal Usage Tracking System (GONUTS), we describe an approach to expand biocuration through crowdsourcing with undergraduates. This multiplies the number of high-quality annotations in international databases, enriches our coverage of the literature on normal gene function, and pushes the field in new directions. From an intercollegiate competition judged by experienced biocurators, Community Assessment of Community Annotation with Ontologies (CACAO), we have contributed nearly 5,000 literature-based annotations. Many of those annotations are to organisms not currently well-represented within GO. Over a 10-year history, our community contributors have spurred changes to the ontology not traditionally covered by professional biocurators. The CACAO principle of relying on community members to participate in and shape the future of biocuration in GO is a powerful and scalable model used to promote the scientific enterprise. It also provides undergraduate students with a unique and enriching introduction to critical reading of primary literature and acquisition of marketable skills.

The Evidence and Conclusion Ontology (ECO) contains terms (classes) that describe types of evidence and assertion methods. ECO terms are used in the process of biocuration to capture the evidence that supports biological assertions (e.g. gene product X has function Y as supported by evidence Z). Capture of this information allows tracking of annotation provenance, establishment of quality control measures and query of evidence. ECO contains over 1500 terms and is in use by many leading biological resources including the Gene Ontology, UniProt and several model organism databases. ECO is continually being expanded and revised based on the needs of the biocuration community. The ontology is freely available for download from GitHub (https://github.com/evidenceontology/) or the project's website (http://evidenceontology.org/). Users can request new terms or changes to existing terms through the project's GitHub site. ECO is released into the public domain under CC0 1.0 Universal.

Self-assembling proteins and protein fragments encoded by the Escherichia coli genome were identified from E. coli K-12 strain MG1655. Libraries of random DNA fragments cloned into a series of [lambda] repressor fusion vectors were subjected to selection for immunity to infection by phage [lambda] Survivors were identified by sequencing the ends of the inserts, and the fused protein sequence was inferred from the known genomic sequence. Four hundred sixty-three nonredundant open reading frame-encoded interacting sequence tags (ISTs) were recovered from sequencing 2,089 candidates. These ISTs, which range from 16 to 794 amino acids in length, were clustered into families of overlapping fragments, identifying potential homotypic interactions encoded by 232 E. coli genes. Repressor fusions identified ISTs from genes in every protein-based functional category, but membrane proteins were underrepresented. The IST-containing genes were enriched for regulatory proteins and for proteins that form higher-order oligomers. Forty-eight (20.7%) homotypic proteins identified by ISTs are predicted to contain coiled coils. Although most of the IST-containing genes are identifiably related to proteins in other bacterial genomes, more than half of the ISTs do not have identifiable homologs in the Protein Data Bank, suggesting that they may include many novel structures. The data are available online at http://oligomers.tamu.edu/.

Regulation of gene expression by many transcription factors is controlled by specific combinations of homo- and heterodimers through a short [Alpha]-helical coiled-coil known as a leucine zipper. The dimer interface of a leucine zipper involves side chains of the residues at the a, d, e, and g positions of the [(abcdefg).sub.n] heptad repeat. To understand the basis for the specificity of dimer formation, we characterized GCN4 leucine zipper mutants with all 16 possible permutations and combinations of isoleucines and asparagines at four a positions in the dimer interface, using a genetic test for the specificity of dimer formation by [Lambda] repressor-leucine zipper fusions. Heterodimers were detected by loss of repressor activity in the presence of a fusion to a dominant-negative mutant form of the DNA-binding domain of repressor. Reconstruction experiments using leucine zippers from GCN4, Jun, Fos, and C/EBP showed that this assay distinguishes pairs that form heterodimers from those that do not. We found that the mutants have novel dimerization specificities determined by the positioning of buried asparagine residues at the a positions. The pattern of buried polar residues could also explain the dimerization specificities of some naturally occurring leucine zippers. The altered specificity mutants described here should be useful for the construction of artificial regulatory circuitry.

The first 6 amino acids ([NH.sub.2]-[Ser.sup.1]-[Thr.sup.2]-[Lys.sup.3]-[Lys.sup.4]-[Lys.sup.5]-[Pro. sup.6]) of bacteriophage [lambda] cI repressor form a flexible arm that wraps around the operator DNA. Homodimeric A repressor has two arms. To determine whether both arms are necessary or only one arm is sufficient for operator binding, we constructed heterodimeric repressors with two, one, or no arms by fusing the DNA binding domain of A repressor to leucine zippers from Fos and Jun. Although only one arm is visible in the cocrystal structure of the N-domain--operator complex, our results indicate that both arms are required for optimal operator binding and normal site discrimination.

A genetic system was developed in Escherichia coli to study leucine zippers with the amino-terminal domain of bacteriophage λ repressor as a reporter for dimerization. This system was used to [...]

Abstract Background Systems biology and functional genomics require genome-wide datasets and resources. Complete sets of cloned open reading frames (ORFs) have been made for about a dozen bacterial species and allow researchers to express and study complete proteomes in a high-throughput fashion. Results We have constructed an open reading frame (ORFeome) collection of 3974 or 94% of the known Escherichia coli K-12 ORFs in Gateway® entry vector pENTR/Zeo. The collection has been used for protein expression and protein interaction studies. For example, we have compared interactions among YgjD, YjeE and YeaZ proteins in E. coli, Streptococcus pneumoniae, and Staphylococcus aureus. We also compare this ORFeome with other Gateway-compatible bacterial ORFeomes and show its utility for comparative functional genomics. Conclusions The E. coli ORFeome provides a useful resource for functional genomics and other areas of protein research in a highly flexible format. Our comparison with other ORFeomes makes comparative analyses straighforward and facilitates direct comparisons of many proteins across many genomes.